Method of producing plants having enhanced transpiration efficiency and plants produced therefrom

ABSTRACT

The present invention provides methods of selecting plants having modified transpiration efficiency using plant ERECTA gene sequences and nucleic acids linked thereto, and to methods of producing plants having modified transpiration efficiency using isolated plant ERECTA gene sequences, in both traditional plant breeding and genetic engineering approaches. The invention further extends to plants produced by the methods described.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding and theproduction of genetically engineered plants. More specifically, theinvention described herein provides genes that are capable of enhancingthe transpiration efficiency of a plant when expressed therein. Thesegenes are particularly useful for the production of plants havingenhanced transpiration efficiency, by both traditional plant breedingand genetic engineering approaches. The invention further extends toplants produced by the methods described herein.

BACKGROUND TO THE INVENTION

1. General

This specification contains nucleotide and amino acid sequenceinformation prepared using PatentIn Version 3.1, presented herein afterthe claims. Each nucleotide sequence is identified in the sequencelisting by the numeric indicator <210> followed by the sequenceidentifier (e.g. <210>1, <210>2, etc). The length and type of sequence(DNA, protein (PRT), etc), and source organism for each nucleotidesequence, are indicated by information provided in the numeric indicatorfields <211>, <212> and <213>, respectively. Nucleotide sequencesreferred to in the specification are defined by the term “SEQ ID NO:”,followed by the sequence identifier (eg. SEQ ID NO: 1 refers to thesequence in the sequence listing designated as <400>1).

The designation of nucleotide residues referred to herein are thoserecommended by the IUPAC-IUB Biochemical Nomenclature Commission,wherein A represents Adenine, C represents Cytosine, G representsGuanine, T represents thymine, Y represents a pyrimidine residue, Rrepresents a purine residue, M represents Adenine or Cytosine, Krepresents Guanine or Thymine, S represents Guanine or Cytosine, Wrepresents Adenine or Thymine, H represents a nucleotide other thanGuanine, B represents a nucleotide other than Adenine, V represents anucleotide other than Thymine, D represents a nucleotide other thanCytosine and N represents any nucleotide residue.

As used herein the term “derived from” shall be taken to indicate that aspecified integer is obtained from a particular source albeit notnecessarily directly from that source.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated step or element orinteger or group of steps or elements or integers but not the exclusionof any other step or element or integer or group of elements orintegers.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purposes ofexemplification only. Functionally equivalent products, compositions andmethods are clearly within the scope of the invention, as describedherein.

2. Description of the Related Art

It is well known that virtually all plants require a certain quantity ofwater for proper growth and development, because CO₂ fixation andphotosynthate assimilation by plants cost water. A significant quantityof water absorbed by plants from the soil returns to the atmosphere viaplant transpiration.

Transpiration efficiency is a measure of the amount of dry matterproduced by a plant per unit of water transpired, or, in other words,carbon gain relative to water lost through transpiration.

For plants having low transpiration efficiency, or when water is inshort supply, the loss of water through transpiration can limit keymetabolic processes associated with plant growth and development. Forexample, during drought, or when plants having low transpirationefficiency are grown in arid and semi-arid environments, plantproductivity as determined by dry matter production or photosyntheticrate, is considerably reduced. Accordingly, the production of plantshaving enhanced water use efficiency or transpiration efficiency ishighly desirable for their adaptation to arid or semi-arid conditions,or to enhance their drought resistance.

The enhancement of water use efficiency or transpiration efficiency byplants is also highly desirable in consideration of global climaticchange and increasing pressure on world water resources. The inefficientutilization of agricultural water is known to impact adversely upon thesupply of navigable water, potable water, and water for industrial orrecreational use. Accordingly, the production of plants having enhancedtranspiration efficiency is highly desirable for reducing the pressureon these water resources. It is also desirable for increasing plantproductivity under well-watered conditions.

By enhancing transpiration efficiency, carbon gain rates are enhancedper unit of water transpired, thereby stimulating plant growth underwell-watered conditions, or alternatively, under mild or severe droughtconditions. This is achieved by enhancing carbon gain more thantranspiration rate, or by reducing the amount of water lost at anyparticular rate of carbon fixation. Those skilled in the art alsoconsider that for a given growth rate plants having enhancedtranspiration efficiency dry out soils more slowly, and use less water,than less efficient near-isogenic plants.

Several chemical as well as environmental pre-treatments have beendescribed for enhancing the ability of plant seedlings to survivedrought, either by reducing transpiration or by reducing the amount ofwater that is actually lost to the atmosphere.

Known environmental treatments largely involve the use of physicalbarriers. Whilst placing a physical barrier over plant stomata is knownto reduce water loss via transpiration, the procedure is not alwaysdesirable or practicable for field-grown crops. For example, physicalbarriers over plant stomata may inhibit certain gas-exchange processesof the plant. It is more desirable to enhance actual transpirationefficiency or water use efficiency of the plant through manipulation ofintrinsic plant function.

Chemical agents are typically the so-called “anti-transpirant” or“anti-desiccant” agents, both of which are applied to the leaves.Anti-transpirants are typically films or metabolic anti-transpirants.

These products form a film on leaves, thereby either blocking stomatalpores, or coating leaf epidermal cells with a water-proof film. Typicalfilm anti-transpirants include waxes, wax-oil emulsions, higheralcohols, silicones, plastics, latexes and resins. For example, Elmore,U.S. Pat. No. 4,645,682 disclosed an anti-transpirant consisting of anaqueous paste wax; Cushman et al., U.S. Pat. Nos. 3,791,839 and3,847,641 also disclosed wax emulsions for controlling transpiration inplants; and Petrucco et al., U.S. Pat. No. 3,826,671, disclosed apolymer composition said to be effective for controlling transpirationin plants.

Metabolic anti-transpirants generally close stomata, thereby reducingthe rate of transpiration. Typical metabolic anti-transpirants includesuccinic acids, phenylmercuric acetate, hydroxysulfonates, the herbicideatrazine, sodium azide, and phenylhydrazones, as well as carbon cyanide.

Compounds having plant growth regulator activity have also been shown tobe useful for reducing transpiration. For example, Bliesner et al., U.S.Pat. No. 4,671,816, disclosed an acetylene compound, said to possessutility for regulating plant growth, whilst Kuznetsov et al. (RussianPatent No. SU 1,282,492;., Russian Patent Application No. SU1,253,559-A1), and Smirnov et al (Russian Patent No. SU 1,098,934)disclosed the use of derivatives of 2-methyl-5-hydroxybenzimidazole, andthe chloride or bromide salts thereof, as anti-transpirant growthregulators. Vichnevetskaia (U.S. Ser. No. 5,589,437 issued Dec. 31,1996) also describe hydroxybenzimidazole derivatives for enhancing thedrought resistance of plants by reducing transpiration. Schulz et al.,U.S. Pat. No. 4,943,315, also disclosed formulations comprising anacetylene and a phenylbenzylurea compound, for reducing transpiration inplants and/or for avoiding impairment to plants caused by heat and dryconditions. Abscisic acid has also been shown to reduce or suppresstranspiration in plants (eg. Helv. Chim. Acta, 71, 931, 1988; J. Org.Chem., 54, 681, 1989; and Japanese Patent Publication No. 184,966/1991).

Metabolic anti-transpirants are costly to produce and often exhibitphytotoxic effects or inhibit plant growth (Kozlowski (1979), In: TreeGrowth and Environmental Stresses (Univ. of Washington Press, Seattleand London)), and are not practically used.

Recent studies have examined alternative methods for enhancingtranspiration efficiency, particularly breeding approaches to selectlines that grow more efficiently under mild drought conditions. Carbonisotope discrimination has been used to identify Arabidopsis ecotypeswith contrasted transpiration efficiencies (Masle et al., In: Stableisotopes and plant carbon-water relations, Acad. Press, Physiol. Ser.,pp 371-386, 1993) and to assist conventional breeding of new plantvarieties in a number of species (Hall et al., Plant Breeding Reviews 4,81-113, 1994) including rice (Farquhar et al., In: Breaking the YieldBarrier, ed KG Cassman, IRRI, 95, 101) and most recently wheat (Rebetzkeet al. Crop Science 42:739-745, 2002).

No single gene has been identified as being capable of enhancingtranspiration efficiency when expressed in planta. Transpirationefficiency may well be multigenic. As a consequence, the genes andsignalling pathways that regulate the photosynthetic and/or stomatalcomponents of the transpiration efficiency mechanism in plants have notbeen identified or characterized.

Moreover, notwithstanding that the effect of down-regulating expressionof the Rubisco gene, or mutation in genes involved in abscisic acid (eg.aba, abi), are known to modify transpiration efficiency to some extentthrough stomatal closure, the consequence of such modifications is notnecessarily specific, resulting in pleiotropic effects.

Arabidopsis thaliana ecotype Landsberg erecta (L-er1) is one of the mostpopular ecotypes and is used widely for both molecular and geneticstudies. It harbors the er1 mutation, which confers a compactinflorescence, blunt fruits, and short petioles. There are a number oferecta mutant alleles. Phenotypic characterization of the mutant allelessuggests a role for the wild type ER gene in regulating plantmorphogenesis, particularly the shapes of organs that originate from theshoot apical meristem. Torii et al. The Plant Cell 8, 735, 1996, showedthat the ER gene encodes a putative receptor protein kinase comprising acytoplasmic protein kinase catalytic domain, a transmembrane region, andan extracellular domain consisting of leucine-rich repeats, which arethought to interact with other macromolecules.

SUMMARY OF THE INVENTION

In work leading up to the present invention, the inventors sought toelucidate the specific genetic determinants of plant transpirationefficiency. In plants, the development of molecular genetic markers,such as, for example, genetic markers that map to a region of the genomeof a crop plant, such as, for example, a region of the rice genome,maize genome, barley genome, sorghum genome, or wheat genome, or aregion of the tomato genome or of any Brassicaceae, assists in theproduction of plants having enhanced transpiration efficiency (Edwardset al., Genetics 116, 113-125, 1987; Paterson et al., Nature 335,721-726, 1988).

The present inventors identified a locus that is linked to the geneticvariation in transpiration efficiency in plants. To elucidate a locusassociated with the transpiration efficiency of plants, the inventorsestablished experimental conditions and sampling procedures to determinethe contribution to total transpiration efficiency of the factorsinfluencing this phenotype, and, more particularly, the geneticcontribution to the total variation in transpiration efficiency. Factorsinfluencing transpiration efficiency include, for example, genotype ofthe plant, environment (eg. temperature, light, humidity, boundary layeraround the leaves, root growth conditions), development (eg. age and/orstage and/or posture of plants that modify gas exchange and/or carbonmetabolism), and seed-specific factors (Masle et al. 1993, op. cit). Thescreens developed by the inventors were also used to survey mutant andwild type populations for variations in transpiration efficiency and toidentify ecotypes having contrasting transpiration efficienciesincluding the parental lines that had been used by Lister and Dean(1993). The transpiration efficiencies of the members of Lister andDean's (1993) Recombinant Inbred Line (RIL) mapping population were thendetermined, and linkage analyses were performed against genetic markersto determine the chromosome regions that are linked to genetic variationin transpiration efficiency, thereby identifying a locus conditioningtranspiration efficiency. Complementation tests, wherein plants weretransformed with a wild-type allele at this locus confirmed thefunctionality of the allele in determining a transpiration efficiencyphenotype.

In one exemplified embodiment of the invention, there is provided alocus associated with transpiration efficiency of A. thaliana, such as,for example the ERECTA locus on A. thaliana chromosome 2, or ahybridization probe which maps to the region between about 46 cM andabout 50.7 cM on chromosome 2 of A. thaliana. In further exemplifiedembodiments, the inventors identified additional ERECTA alleles orerecta alleles in A. thaliana, rice, sorghum, wheat and maize which arestructurally related to this primary A. thaliana ERECTA or erectaallele. Based upon the large number of ERECTA/erecta alleles describedherein, the present invention clearly extends to any homologs of the A.thaliana ERECTA locus from other plant species to those specificallyexemplified, and particularly when those homologs are identified usingthe methods described herein.

Accordingly, one aspect of the invention provides a genetic marker orlocus associated with the genetic variation in transpiration efficiencyof a plant, wherein said locus comprises a nucleotide sequence linkedgenetically to an ERECTA locus in the genome of the plant. The locus orgenetic marker is useful for determining transpiration efficiency of aplant.

As used herein, the terms “genetically linked” and “map to” shall betaken to refer to a sufficient genetic proximity between a linkednucleic acid comprising a gene, allele, marker or other nucleotidesequence and nucleic acid comprising all or part of an ERECTA locus topermit said linked nucleic acid to be useful for determining thepresence of a particular allele of said ERECTA locus in the genome of aplant. Those skilled in the art will be aware that for such linkednucleic acid to be used in this manner, it must be sufficiently close tosaid locus not to be in linkage disequilibrium or to have a highrecombination frequency between said linked nucleic acid and said locus.Preferably, the linked nucleic acid and the locus are less than about 25cM apart, more preferably less than about 10 cM apart, even morepreferably less than about 5 cM apart, still even more preferably lessthan about 3 cM apart and still even more preferably less than about 1cM apart.

In a preferred embodiment the present invention provides an isolatednucleic acid associated with the genetic variation in transpirationefficiency of a plant, said nucleic acid comprising a nucleotidesequence selected from the group consisting of:

-   -   (a) the sequence of an ERECTA genomic gene or the 5′-UTR or        3′-UTR or protein-encoding region or an intron region thereof;    -   (b) the sequence of an allelic variant of (a) or the 5′-UTR or        3′-UTR or protein-encoding region or an intron region of said        allelic variant;    -   (c) the sequence of a fragment of (a) or (b) that hybridizes        specifically to nucleic acid (eg., RNA or DNA) from a plant        under at least low stringency hybridization conditions; and    -   (d) a sequence that is complementary to (a) or (b) or (c).

In a particularly preferred embodiment, the present invention providesan isolated ERECTA gene from wheat comprising a nucleotide sequenceselected from the group consisting of:

(i) the sequence set forth in SEQ ID NO: 19;

(ii) a sequence encoding the amino acid sequence set forth in SEQ ID NO:20; and

(iii) a sequence that is complementary to (i) or (ii).

In an alternative embodiment, the present invention provides an isolatedERECTA gene from maize comprising a nucleotide sequence selected fromthe group consisting of:

(i) the sequence set forth in SEQ ID NO: 44;

(ii) a sequence encoding the amino acid sequence set forth in SEQ ID NO:45; and

(iii) a sequence that is complementary to (i) or (ii).

In another alternative embodiment, the present invention provides anisolated ERECTA gene from rice comprising a nucleotide sequence selectedfrom the group consisting of:

(i) the sequence set forth in SEQ ID NO: 3;

(ii) a sequence encoding the amino acid sequence set forth in SEQ ID NO:4; and

(iii) a sequence that is complementary to (i) or (ii).

In another alternative embodiment, the present invention provides anisolated ERECTA gene from A. thatliana comprising a nucleotide sequenceselected from the group consisting of:

(i) the sequence set forth in SEQ ID NO: 1;

(ii) a sequence encoding the amino acid sequence set forth in SEQ ID NO:2; and

(iv) a sequence that is complementary to (i) or (ii).

In another alternative embodiment, the present invention provides anisolated ERECTA gene from A. thatliana comprising a nucleotide sequenceselected from the group consisting of:

(i) the sequence set forth in SEQ ID NO: 7;

(ii) a sequence encoding the amino acid sequence set forth in SEQ ID NO:8; and

(v) a sequence that is complementary to (i) or (ii).

In another alternative embodiment, the present invention provides anisolated ERECTA gene from A. thatliana comprising a nucleotide sequenceselected from the group consisting of:

(i) the sequence set forth in SEQ ID NO: 9;

(ii) a sequence encoding the amino acid sequence set forth in SEQ ID NO:10; and

(vi) a sequence that is complementary to (i) or (ii).

In yet another alternative embodiment, the present invention provides anisolated ERECTA gene from sorghum comprising a nucleotide sequenceselected from the group consisting of:

(i) the sequence set forth in SEQ ID NO: 5;

(ii) a sequence encoding the amino acid sequence set forth in SEQ ID NO:6; and

(vii) a sequence that is complementary to (i) or (ii).

Notwithstanding that an ERECTA or erecta structural gene or genomic geneor the protein encoding region thereof is particularly useful forbreeding and/or mapping purposes, this aspect of the present inventionis not to be limited to the ERECTA or erecta structural or genomic geneor the protein-encoding region thereof. As exemplified herein, theprimary A. thaliana ERECTA locus can be determined using any linkednucleic acid that maps to a region in the chromosome at a geneticdistance of up to about 3 cM from the ERECTA or erecta allele. Theskilled artisan will readily be able to utilize similar probes toidentify linkage to an ERECTA or erecta allele in any other plantspecies, based upon the teaching provided herein that the ERECTA orerecta allele is linked to the transpiration efficiency phenotype ofplants.

Preferably, all or part of the locus associated with the transpirationefficiency phenotype in a plant (ie., nucleic acid genetically linked tothe ERECTA or erecta structural or genomic gene) is provided asrecombinant or isolated nucleic acid, such as, for example, in the formof a gene construct (eg. a recombinant plasmid or cosmid), to facilitategermplasm screening.

The ERECTA locus or a gene that is linked to the ERECTA locus isparticularly useful in a breeding program, to predict the transpirationefficiency of a plant, or alternatively, as a selective breeding markerto select plants having enhanced transpiration efficiency. Once mapped,marker-assisted selection (MAS) is used to introduce the ERECTA locus ormarkers linked thereto into a wide variety of populations. MAS has theadvantage of reducing the breeding population size required, and theneed for continuous recurrent testing of progeny, and the time requiredto develop a superior line.

Accordingly, a further aspect of the present invention provides a methodof selecting a plant having enhanced transpiration efficiency,comprising detecting a genetic marker for transpiration efficiency whichmarker comprises a nucleotide sequence linked genetically to an ERECTAlocus in the genome of the plant and selecting a plant that comprises orexpresses the genetic marker, preferably wherein the genetic markercomprises an ERECTA allele or erecta allele, or a protein-encodingportion thereof, or alternatively, wherein the genetic marker comprisesa nucleotide sequence having at least about 55% overall sequenceidentity to at least about 20 nucleotides in length of any one of SEQ IDNos: 1, 3, 5, 7, 9, 11 to 19 or 21 to 44 or a complementary sequencethereto, including a nucleotide sequence selected from the groupconsisting of:

-   -   (a) a sequence having at least about 55% identity to a sequence        selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:        3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ        ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID        NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 SEQ ID NO:        21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,        SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ        ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 SEQ ID NO: 33, SEQ ID        NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:        38; SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42,        SEQ ID NO: 43, SEQ ID NO: 44;    -   (b) a sequence encoding an amino acid sequence having at least        about 55% identity to an amino acid sequence selected from the        group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 20 and        SEQ ID NO: 45; and    -   (c) a sequence complementary to (a) or (b).

In an alternative embodiment, the invention provides a method ofselecting a plant having enhanced transpiration efficiency, comprising:

-   -   (a) screening mutant or near-isogenic or recombinant inbred        lines of plants to segregate alleles at an ERECTA locus;    -   (b) identifying a polymorphic marker linked to said ERECTA        locus; and    -   (c) selecting a plant that comprises or expresses the marker.

The data exemplified herein for A. thaliana or rice can clearly beextrapolated to other plant species. For example, the evidence providedherein for the role of the A. thaliana ERECTA allele in determining thetranspiration efficiency phenotype in those plant species has permittedthe elucidation of a wide range of homologous ERECTA alleles in otherplant species, in particular wheat, rice, sorghum and maize, that arealso likely to determine the transpiration efficiency phenotype in thoseplants. In accordance with this embodiment, the present inventionprovides a method of selecting a plant having enhanced transpirationefficiency, comprising selecting a plant that comprises or expresses afunctionally equivalent homolog of a protein-encoding region of theERECTA gene of A. thaliana, maize, wheat, sorghum or rice.

In a preferred embodiment, the invention provides a method of selectinga plant having enhanced transpiration efficiency, comprising:

-   -   (a) identifying a locus on the Arabidopsis chromosome 2 (46-50.7        cM) or rice chromosome 6 associated with genetic variation in        transpiration efficiency in a plant;    -   (b) identifying nucleic acid in a different plant species that        comprises a nucleotide sequence having at least about 55%        identity to the sequence of the locus at (a); and    -   (c) selecting a plant that comprises or expresses the identified        nucleic acid at (b).

In a further preferred embodiment, this aspect of the invention providesa method of selecting a plant having enhanced transpiration efficiency,comprising:

-   -   (a) identifying a locus on the Arabidopsis chromosome 2 (46-50.7        cM) or rice chromosome 6 associated with genetic variation in        transpiration efficiency in a plant;    -   (b) determining the nucleotide sequence of the identified locus;    -   (c) identifying nucleic acid of a plant species other than A.        thaliana or rice that comprises a nucleotide sequence having at        least about 55% identity to the sequence of the locus at (a);        and    -   (d) selecting a plant that comprises or expresses the identified        nucleic acid at (b).

Preferably, the selected plant according to any one or more of thepreceding embodiments is Arabidopsis thaliana, rice, sorghum, wheat ormaize, however other species are not excluded.

Preferably, the subject selection method comprises linking thetranspiration efficiency phenotype of the plant to the expression of themarker in the plant, or alternatively, linking a structural polymorphismin DNA to a transpiration efficiency phenotype in the plant, eg., by aprocess comprising detecting a restriction fragment length polymorphism(RFLP), amplified fragment length polymorphism (AFLP), single strandchain polymorphism (SSCP) or microsatellite analysis. As will be knownto the skilled artisan, a nucleic acid probe or primer of at least about20 nucleotides in length from any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11to 19 or 21 to 44 or a complementary sequence thereto can be hybridizedto genomic DNA from the plant, and the hybridization detected using adetection means, thereby identifying the polymorphism.

It is clearly preferred that the selected plant has enhancedtranspiration efficiency compared to a near-isogenic plant that does notcomprise or express the genetic marker.

As exemplified herein, the inventors also identified specific genes oralleles that are linked to the ERECTA locus of A. thaliana, and rice anddetermined the transpiration efficiencies of those plants. Moreparticularly, the transpiration efficiencies of near-isogenic lines,each carrying a mutation within an ERECTA locus, and a correlationbetween transpiration efficiency phenotype and ERECTA expression or genecopy number are determined, thereby providing the genetic contributionof genes or alleles at the ERECTA locus to transpiration efficiency.This analysis permits an assessment of the genetic contribution ofparticular alleles to transpiration efficiency, thereby determiningallelic variants that are linked to a particular transpirationefficiency. Thus, the elucidation of the ERECTA locus for transpirationefficiency in plants facilitates the fine mapping and determination ofallelic variants that modulate transpiration efficiency. The methodsdescribed herein can be applied to an assessment of the contribution ofspecific alleles to the transpiration efficiency phenotype for any plantspecies that is amenable to mutagenesis such as, for example, bytransposon mutagenesis, irradiation, or chemical means. As will be knownto the skilled artisan many crop species, such as, maize, wheat, andrice, are amenable to such mutagenesis.

Accordingly, a third aspect of the invention provides a method ofidentifying a gene that determines the transpiration efficiency of aplant comprising:

-   -   (a) identifying a locus associated with genetic variation in        transpiration efficiency in a plant;    -   (b) identifying a gene or allele that is linked to said locus,        wherein said gene or allele is a candidate gene or allele for        determining the transpiration efficiency of a plant; and    -   (c) determining the transpiration efficiencies of a panel of        plants, wherein not all members of said panel comprise or        express said gene or allele, and wherein variation in        transpiration efficiency between the members of said panel        indicates that said gene is involved in determining        transpiration efficiency.

In another embodiment, the method comprises:

-   -   (a) identifying a locus associated with genetic variation in        transpiration efficiency in a plant;    -   (b) identifying multiple alleles of a gene that is linked to        said locus, wherein said gene is a candidate gene involved for        determining the transpiration efficiency of a plant; and    -   (c) determining the transpiration efficiencies of a panel of        plants, wherein each member of said panel comprises, and        preferably expresses, at least one of said multiple alleles,        wherein variation in transpiration efficiency between the        members of said panel indicates that said gene is involved in        determining transpiration efficiency.

Preferably, the identified gene or allele identified by the methoddescribed in the preceding paragraph is an ERECTA allele, or an erectaallele, from a plant selected from the group consisting of A. thaliana,sorghum, rice, maize and wheat, or a homolog thereof.

The identified gene or allele, including any homologs from a plant otherthan A. thaliana, such as, for example, the wild-type ERECTA allele or ahomolog thereof, is useful for the production of novel plants. Suchplants are produced, for example, using recombinant techniques, ortraditional plant breeding approaches such as introgression.

Accordingly, a still further aspect of the present invention provides amethod of modulating (i.e., enhancing or reducing) the transpirationefficiency of a plant comprising ectopically expressing in a plant anisolated ERECTA gene or an alleic variant thereof or theprotein-encoding region of said ERECTA gene or said allelic variant. Ina particularly preferred embodiment, the invention provides a method ofenhancing the transpiration efficiency of a plant comprisingintrogressing into said plant a nucleic acid comprising a nucleotidesequence that is homologous to a protein-encoding region of a gene of A.thaliana that maps to the ERECTA locus on chromosome 2.

A further embodiment of the invention provides a method of modulatingthe transpiration efficiency of a plant comprising introducing (eg., byclassical breeding, introgression or recombinant means), and preferablyexpressing therein, an isolated ERECTA gene or an allelic variantthereof or the protein-encoding region thereof to a plant and selectinga plant having a different transpiration efficiency compared to anear-isogenic plant that does not comprise the introduced ERECTA gene orallelic variant or protein-encoding region. Preferably, the ERECTA geneor allelic variant or protein-encoding region comprises a nucleotidesequence selected from the group consisting of:

-   -   (a) a sequence having at least about 55% identity to a sequence        selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:        3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ        ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID        NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 SEQ ID NO:        21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,        SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ        ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 SEQ ID NO: 33, SEQ ID        NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:        38; SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42,        SEQ ID NO: 43 and SEQ ID NO: 44; and    -   (b) a sequence encoding an amino acid sequence having at least        about 55% identity to an amino acid sequence selected from the        group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 20 and        SEQ ID NO: 45.

The plant into which the gene etc is introduced is preferably selectedfrom the group consisting of Arabidopsis thaliana, rice, sorghum, wheatand maize. As will be apparent from the present disclosure, thetranspiration efficiency is enhanced as a consequence of the ectopicexpression of an ERECTA allele or the protein-encoding region thereof inthe plant. In contrast, the transpiration efficiency is reduced as aconsequence of reduced expression of an ERECTA allele in the plant (eg.,by expression of antisense RNA or RNAi or other inhibitory RNA).

A further aspect of the invention provides for the use of an isolatedERECTA gene or an allelic variant thereof or the protein-encoding regionof said ERECTA gene or said allelic variant in the preparation of a geneconstruct for modulating (ie., enhancing or reducing) the transpirationefficiency of a plant. For example, expression of ERECTA protein in theplant can be modified by ectopic expression of an ERECTA allele in theplant, or alternatively, by reducing endogenous ERECTA expression usingan inhibitory RNA (eg, antisense or RNAi).

A fifth aspect of the present invention provides a plant having enhancedtranspiration efficiency, wherein said plant is produced by a methoddescribed herein.

Plants that have enhanced transpiration efficiency show increased levelsof growth under normal growth conditions, thereby increasing theirbiomass. Accordingly, a further aspect of the present invention providesa method of increasing the biomass of a plant comprising enhancing thelevel of expression of an ERECTA gene or allelic variant thereof orprotein coding region thereof in said plant.

In one embodiment, the method further includes the step of selecting aplant that has an increased biomass when compared to an unmodifiedplant. Methods of determining the biomass of a plant are well known tothose skilled in the art and/or described herein.

In one embodiment, the level of expression is enhanced by geneticmodification of a control sequence, for example a promoter sequence;associated with the ERECTA gene or allelic variant thereof.

In another embodiment, the level of expression is enhanced byintroducing (eg., by classical breeding, introgression or recombinantmeans) an ERECTA gene or allelic variant thereof or the protein encodingregion thereof to a plant. Preferably, the ERECTA gene or allelicvariant or protein-encoding region comprises a nucleotide sequenceselected from the group consisting of:

a sequence having at least about 55% identity to a sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19 SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 SEQ ID NO: 33,SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:38; SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ IDNO: 43 and SEQ ID NO: 44; and

a sequence encoding an amino acid sequence having at least about 55%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 20 and SEQ ID NO: 45.

The plant into which the gene etc is introduced is preferably selectedfrom the group consisting of Arabidopsis thaliana, rice, sorghum, wheatand maize.

A further aspect of the present invention provides a method ofincreasing the resistance of a plant to an environmental stresscomprising enhancing the level of expression of an ERECTA gene orallelic variant thereof or protein coding region thereof in said plant.

As used herein the term “environmental stress” shall be taken in itsbroadest context to mean one or more environmental conditions thatreduce the ability of a plant to grow, survive and/or produceseed/grain. In one embodiment, an environmental stress that affects theability for a plant to grow, survive and/or produce seed/grain is acondition selected from the group consisting of increased or decreasedCO₂ levels, increased or decreased temperature, increased or decreasedrainfall, increased or decreased humidity, increased salt levels in thesoil, increased soil strength and compaction and drought.

In one embodiment, the method further includes the step of selecting aplant that has an altered resistance to an environmental stress whencompared to an unmodified plant is selected. Methods of determining theresistance of a plant to environmental stress are well known to thoseskilled in the art and/or described herein.

In one embodiment, the level of expression is enhanced by geneticmodification of a control sequence, for example a promoter sequence,associated with the ERECTA gene or allelic variant thereof.

In another embodiment, the level of expression is enhanced byintroducing (eg., by classical breeding, introgression or recombinantmeans) an ERECTA gene or allelic variant thereof or the protein encodingregion thereof to a plant. Preferably, the ERECTA gene or allelicvariant or protein-encoding region comprises a nucleotide sequenceselected from the group consisting of:

a sequence having at least about 55% identity to a sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19 SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 SEQ ID NO: 33,SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:38; SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ IDNO: 43 and SEQ ID NO: 44; and

a sequence encoding an amino acid sequence having at least about 55%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 20 and SEQ ID NO: 45.

The plant into which the gene etc is introduced is preferably selectedfrom the group consisting of Arabidopsis thaliana, rice, sorghum, wheatand maize.

A further aspect of the present invention provides a plant havingincreased resistance to environmental stress, wherein said plant isproduced by a method described herein.

Both temperature and available moisture have been shown to dramaticallyinfluence pollination and grain/seed development, processes known asseed-set and grain-filling. Accordingly, a method that produces a plantthat is resistant to environmental stress, ie a plant that has increasedtranspiration efficiency, results in increased or more efficientgrain-filling and greater seed number. As ERECTA is expressed duringflowering or pod development this gene or an allelic variant thereof isuseful for increasing grain-filling in a plant.

Accordingly, a further aspect of the present invention provides a methodof increasing seed or grain weight in a plant comprising enhancing thelevel of expression of an ERECTA gene or allelic variant thereof orprotein coding region thereof in said plant.

In one embodiment, the method further includes the step of selecting aplant that has increased seed or grain weight when compared to anunmodified plant is selected. Methods of determining seed or grainweight are well known to those skilled in the art and/or describedherein.

In one embodiment, the level of expression is enhanced by geneticmodification of a control sequence, for example a promoter sequence,associated with the ERECTA gene or allelic variant thereof.

In another embodiment, the level of expression is enhanced byintroducing (eg., by classical breeding, introgression or recombinantmeans) an ERECTA gene or allelic variant thereof or the protein encodingregion thereof to a plant. Preferably, the ERECTA gene or allelicvariant or protein-encoding region comprises a nucleotide sequenceselected from the group consisting of:

a sequence having at least about 55% identity to a sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19 SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 SEQ ID NO: 33,SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:38; SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ IDNO: 43 and SEQ ID NO: 44; and

a sequence encoding an amino acid sequence having at least about 55%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 20 and SEQ ID NO: 45.

The plant into which the gene etc is introduced is preferably selectedfrom the group consisting of Arabidopsis thaliana, rice, sorghum, wheatand maize.

A further aspect of the present invention provides a plant havingincreased seed or grain weight, wherein said plant is produced by amethod described herein.

A still further aspect of the present invention provides a method ofmodulating the number of seeds produced by a plant comprising enhancingthe level of expression of an ERECTA gene or allelic variant thereof insaid plant.

In one embodiment, the method further includes the step of selecting aplant that has an increased number of seeds when compared to anunmodified plant is selected. Methods of determining seed or grainnumber are well known to those skilled in the art and/or describedherein.

In one embodiment, the level of expression is enhanced by geneticmodification of a control sequence, for example a promoter sequence,associated with the ERECTA gene or allelic variant thereof.

In another embodiment, the level of expression is enhanced byintroducing (eg., by classical breeding, introgression or recombinantmeans) an ERECTA gene or allelic variant thereof or the protein encodingregion thereof to a plant. Preferably, the ERECTA gene or allelicvariant or protein-encoding region comprises a nucleotide sequenceselected from the group consisting of:

a sequence having at least about 55% identity to a sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19 SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 SEQ ID NO: 33,SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:38; SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ IDNO: 43 and SEQ ID NO: 44; and

a sequence encoding an amino acid sequence having at least about 55%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 20 and SEQ ID NO: 45.

The plant into which the gene etc is introduced is preferably selectedfrom the group consisting of Arabidopsis thaliana, rice, sorghum, wheatand maize.

A further aspect of the present invention provides a plant having anincreased number of seeds, wherein said plant is produced by a methoddescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a graphical representation showing the CO₂ assimilationrates (μmol C m² s⁻¹) of several genotypes of A. thaliana. Measurementswere completed on rosette leaves during bolting and flowering stages.Plants were grown on fertilised soil. The genotypes of plants areindicated on the x-axis, and CO₂ assimilation rates indicated on theordinate. Col indicates a genetic background of the ecotype Columbia. Ldindicates a genetic background of the ecotype Landsberg. Plantsexpressing wild type ERECTA alleles were either in a Col (Col4-ER) or Ld(Ld-ER) background. Plants that were homozygous for a mutant er allelewere either in a Ld background (Ld-er1) or in a Col background(Col-er105 or Col-er2 (line 3401 at NASC, also named Col-er106 by Toriiand collaborators ( see Lease et al. 2001, New Phytologist,151:133-143)). Plants designated as F1 (Col-ER×Ld-er) were heterozygousER/er1. Data indicate that, in a Col background, the er105 mutationleads to reduced CO₂ assimilation rate, whilst the er1 mutation enhancesCO₂ assimilation rate in a Ld background.

FIG. 1 b is a graphical representation showing the stomatal conductance(mol H₂0 m² s⁻¹) of several genotypes of A. thaliana (same plants asFIG. 1 a). The genotypes of plants are indicated on the x-axis and arethe same as described in the legend to FIG. 1 a. Stomatal conductancesare indicated on the ordinate. Data indicate that, in a Col background,the er2/er106 mutation significantly enhances stomatal conductance,whilst the er1 mutation significantly enhances stomatal conductance in aLd background.

FIG. 1 c is a graphical representation showing the transpirationefficiency of (mmol C mol H₂0⁻¹) of several genotypes of A. thaliana, asdetermined by the ratio of CO₂ assimilation rate to stomatalconductance. The genotypes of plants are indicated on the x-axis and arethe same as described in the legend to FIG. 1 a. Transpirationefficiency is indicated on the ordinate. Data indicate thattranspiration efficiency is enhanced in plants expressing a wild type ERallele relative to a mutant er allele, in both Ld and Col backgrounds.The lowest transpiration efficiency was observed for plants that arehomozygous for the er105 allele (ie. Col-er 105), consistent with thefact that this allele inhibits ERECTA expression. From the data in FIG.1 a-1 c, it is apparent that the lower transpiration efficiency ofplants expressing the er105 allele is largely due to a reduced CO₂fixation rate, whereas for both the er2/er106 and er1 alleles, reducedtranspiration efficiency is largely due to enhanced stomatalconductance. The transpiration efficiency of the F1 heterozygote plantwas intermediate between the transpiration efficiencies of its parents,suggesting codominance of these alleles. The F1, however, had atranspiration efficiency closer to that of the pollen donor parent,Ld-er1.

FIG. 2 a is a graphical representation showing the stomatal densities(Number of stomata mm⁻² leaf) for several genotypes of A. thaliana inthree independent experiments. The genetic backgrounds of plants areindicated on the x-axis (Col, Columbia; Ld, Landsberg), and stomataldensities are indicated on the ordinate. Plant genotypes are indicatedat the top of each bar, as follows: plants expressing wild type ERECTAalleles in a Col background were Col4ER or Col1ER (hatched bars); plantsexpressing wild type ERECTA alleles in a Ld background were ER (openbars); plants expressing mutant erecta alleles in a Col background wereeither er105 or er2/106 (Col filled boxes); and plants expressing themutant er1 allele in a Ld background were er1 (Ld filled boxes). Columnsdesignated a,b are data from two experiments where plants were grown insoil in the absence of fertiliser. The set of columns at the right ofthe figure are from a third experiment where the same plants were grownin soil comprising fertiliser. Data indicate that, in a Col background,the er105 mutation and er2/106 mutation enhances stomatal density, whichin part accounts for the enhanced stomatal conductances and reducedtranspiration efficiencies of plants expressing these alleles (FIGS. 1 band 1 c). The general effect of these alleles is not dependent on thenutrient status of the soil. In contrast, the er1 allele only enhancedstomatal density of Ld plants when fertiliser was absent, suggestingthat in this ecotype enhanced stomatal aperture accounted for theenhanced stomatal conductances and reduced transpiration efficienciesmeasured in the er1 mutant under ample nutrient supply (FIGS. 1 b, 1 c).The er1 mutation therefore affects both stomatal aperture and stomataldensity but the relative contributions of these effects to enhancedstomatal conductance per unit leaf area depend on environmemtal factorsand plant nutrient status, and on genetic background.

FIG. 2 b is a graphical representation showing the epidermal cell size(surface area, μm²) for several genotypes of A. thaliana in threeindependent experiments. The genetic backgrounds and genotypes of plantsare indicated on the x-axis and at the tops of each column,respectively, as in the legend to FIG. 2 a. The ordinate indicatesepidermal cell size. Columns designated a,b are data from twoexperiments where plants were grown in soil in the absence offertiliser. The set of columns at the right of the figure are from athird experiment where the same plants were grown in soil comprisingfertiliser. Data indicate that, in a Col background, the er105 mutationand er2/er106 mutation significantly reduce epidermal cell size ieincrease the number of epidermal cells per unit leaf area. This revealsthat the ER gene has effects on leaf histogenesis which, beyond theirconsequences on stomatal densities, may also directly affect leafcapacity for photosynthesis and therefore transpiration efficiency,(FIGS. 1 b and 1 c). The general effects of these alleles are notdependent on the nutrient status of the soil. In contrast, in a Ldbackground, the er1 allele reduced epidermal cell size only whenfertiliser was absent.

FIG. 2 c is a graphical representation showing the stomatal index forseveral genotypes of A. thaliana in three independent experiments. Thegenetic backgrounds and genotypes of plants are indicated on the x-axisand at the tops of each column, respectively, as in the legend to FIG. 2a. The ordinate indicates stomatal index, as determined from the ratioof stomatal density to epidermal cell density. Columns designated a,bare data from two experiments where plants were grown in soil in theabsence of fertiliser. The set of columns at the right of the figure arefrom a third experiment where the same plants were grown in soilcomprising fertiliser. Data indicate that the er mutations tested do notsignificantly modify stomatal index in Col background (because increasesin stomatal density are correlated to increases in epidermal cellnumbers in the Col mutant plants) but does so in Landsberg background.Accordingly, the ER gene does appear to directly modify stomataldevelopment per se. Taken together FIGS. 2 a-c therefore show that theERECTA gene has two types of effects on leaf stomatal conductance: a)developmental, b) biophysical and/or biochemical. The expression ofthese effects and impact on transpiration rate vary with geneticbackground, suggesting interactions with other genes that arepolymorphic between the Col and Ld ecotypes, and also with nutrientstatus.

FIG. 3 is a graphical representation showing carbon isotope composition(y-axis; in per mil, for vegetative rosettes) for 7 differentexperimental runs (numbers 1-7) carried out under growth cabinetconditions and glasshouse conditions. For each run, the left-hand sidebar shows the mean value of carbon isotope composition for linescarrying the ERECTA allele, while the right-hand side bar shows the meanvalue across lines with the erecta allele. In all cases, δ¹³C isotopiccomposition values for the er-lines are more negative then those for ERlines, indicative of lower transpiration efficiencies.

FIG. 4 a is a graphical representation showing ERECTA gene copy numberand expression levels in transgenic T2 A. thaliana plants homozygous foran ER transgene. These lines were generated by transforming theCol-er2/106 mutant with the wild type ER gene under the 35S promoter.Effective transformation was ascertained and ERECTA expression levelswere quantified in several independent transformants using real-timequantitative PCR (ABI PRISM 7700, Sequence Detection System UserBulletin #2. 1997). Copy number (y-axis) is indicated as a function ofthe plant line, following normalisation of ERECTA relative to the copynumber of a control gene (18S ribosomal RNA gene). The expression of the18S rRNA gene was shown independently not to be affected by changes inER expression. Line 143 is null control (no insert). Lines 145, 165, 169and 279 are transformed lines carrying the ERECTA allele. All ERtransgenic lines, except line 145, show increased mRNA copy number: from4 to 9.5 fold increase compared with the null control.

FIG. 4 b is a graphical representation showing ERECTA gene copy numberand expression levels in transgenic T2 A. thaliana plants homozygous foran ER transgene, and generated by transformation of the Col-er105mutant. Effective transformation was ascertained and ERECTA expressionlevels were quantified in several independent transformants usingreal-time quantitative PCR (ABI PRISM 7700, Sequence Detection SystemUser Bulletin #2. 1997). Copy number (y-axis) is indicated as a functionof the plant line, following normalisation of ERECTA relative to thecopy number of a control gene (18S ribosomal RNA gene). The expressionof the 18S rRNA gene was shown independently not to be affected bychanges in ER expression. Line 18 is a null control line (no ER insert,ie similar to Col-er105). Lines 8, 19, 29 and 61 are transgenic linescarrying the ERECTA allele. All ER transgenic lines show increased mRNAcopy number: from 10 to 170 fold increase compared with the nullcontrol.

FIG. 4 c is a graphical representation showing ERECTA gene copy numberand expression levels in Col and Ld ER ecotypes and in one Ld-ERtransgenic line (3-7K) generated by transformation of the Ld-er1 ecotype(NW20) with the ER wild type gene under control of the 35S promoter.Effective transformation was ascertained and ERECTA expression levelswere quantified in several independent transformants using real-timequantitative PCR (ABI PRISM 7700, Sequence Detection System UserBulletin #2. 1997). Copy number (y-axis) is indicated as a function ofthe plant line, following normalisation of ERECTA relative to the copynumber of a control gene (18S ribosomal RNA gene). The expression of the18S rRNA gene was shown independently not to be affected by changes inER expression. Lines 933, 1093 and 3176 are the non-transformedColumbia-ERECTA ecoptypes Col-4, Col-0 and Col-1. Line 105c is aCol-er105 line (knockout for ER), used for generating transgenic linesshown in FIG. 4 b. Lines labelled 2c and 3401 on the X-axis describeCol-er2/106 (2 batches of seeds, used for generating transgenic linesshown in FIG. 4 a). Line NW20 is Ld-er1. Line 3-7K is a Ld-ERtransformant, obtained from transformation of Ld-er1 with the ERECTAallele. Line 3177 is the Ld-ER ecotype, near-isogenic to NW20.

FIG. 5 a is a graphical representation of a first experiment showingcopy number of the mRNA transcription product of the rice ERECTA gene invarious plant organs/parts, cv Nipponbare. L=mature leaf blades;YL=young expanding leaves, still enclosed in sheaths of older leaves;R=mature root; YR=young root; SH=sheaths; INF=unfolded young paniclestill enclosed in sheaths; O7: young panicles. Rice ERECTA mRNA copynumbers were determined by quantitative real-time PCR, with 18S mRNA asinternal control gene for normalization of results. The values on they-axis describe fold increases of rice ERECTA mRNA in various partscompared to the L sample (mature leaves) set to a value of 1 fornormalization. Data show a similar expression pattern as the ERECTA genein Arabidopsis (see Torii et al. 1996) ie preferential expression inyoung meristematic tissues, especially in reproductive organs.

FIG. 5 b is a graphical representation of a second experiment showingcopy number of the mRNA transcription product of the rice ERECTA gene invarious plant organs/parts. L=mature leaf blades; YL=young expandingleaves, still enclosed in sheaths of older leaves; R=mature root;YR=young root; SH=sheaths; INF=unfolded young panicle still enclosed insheaths; O7: young panicles. Rice ERECTA mRNA copy numbers weredetermined by quantitative real-time PCR, with 18S mRNA as internalcontrol gene for normalization of results. The values on the y-axisdescribe fold increases of rice ERECTA mRNA in various parts compared tothe L sample (mature leaves) set to a value of 1 for normalization. Dataconfirm those shown in FIG. 5 a.

FIG. 6 is a graphical representation showing leaf transpirationefficiency (mmol C mol H₂O⁻¹, FIG. 6 a), calculated from the directmeasurements of leaf CO₂ assimilation rate (μmol C m⁻² s⁻¹, FIG. 6 b)and stomatal conductance (mol H₂O m⁻² s⁻¹, FIG. 6 c) by gas exchangetechniques, under 350 ppm CO₂ (ie same as ambient [CO₂] during seedlinggrowth; left hand bar in each pair of bars) and 500 ppm CO₂ (right handbar in each pair of bars), for Ld-er1, and two Ld_ER lines: lineT2(+ER), a T2 transgenic line homozygous for an ER transgene in theLd-er1 background and line 3177, an ER ecotype near-isogenic to Ld-er1(NASC Stock Centre information). Genotypes are shown at the bottom ofthe figure. Leaf temperature during measurements was controlled at 22°C., leaf to air vapour pressure deficit at around 8 mb.

FIG. 7 is a graphical representation showing leaf transpirationefficiency (mmol C mol H₂O⁻¹, FIG. 7 a), calculated from the directmeasurements of leaf CO₂ assimilation rate (μmol C m⁻² s⁻¹, FIG. 7 b)and stomatal conductance (mol H₂O m⁻² s⁻¹, FIG. 7 c) by gas exchangetechniques, under 350 ppm CO₂ (ie same as ambient [CO₂] during seedlinggrowth; left hand bar in each pair of bars) and 500 ppm CO₂ (right handbar in each pair of bars), for 4 genotypes: Col4 (ER) (left hand pair),Ld (er1) (right hand pair) and their F₁ progeny (middle two pairs).Genotypes are shown at the bottom of the figure.

FIG. 8 is a graphical representation showing stomatal conductance andepidermal anatomy at 350 ppm CO₂ in the genotypes described in FIGS. 6and 7 and shown at the bottom of the figure. The insertion of ERtransgene (line T2+ER) caused a decreased in stomatal conductancecompared to the Ld-er1 line (FIG. 8 a), which was in part due to adecrease in stomatal density (see FIG. 8 c). These two effects againindicate complementation. Together FIGS. 8 b and 8 c show that thedecrease in stomatal density is relatively more important than that inepidermal cell density, indicating an effect of the transgene onepidermis development.

FIG. 9 is a graphical representation showing a comparison of stomataldensity and epidermal cell area in a range of Col er lines carryingmutations in the ER gene (bars 1 to 8 FIG. 9 a; bar 1 to 7 in FIG. 9 b,mutants er105, er106, 108, 111, 114, 116, 117, as described in Lease etal. 2001; a gift from Dr Keiko Torii) and in Col-ER wild type ecotypes(bars 9-11 or 8-10 in FIGS. 9 a and 9 b, respectively: Col0, backgroundecotype for these mutants; Cl1, Col4 (ColER parental line for QTLanalysis of Lister and Dean's RILs), two Ld_er1 lines (NW20 and CS20,bars 12&13 and 11&12 in FIGS. 9 a and 9 b respectively, two very similarlines according to NASC; NW20 is the other parental line for Lister andDean's RILs) and finally line T2+ER, a transgenic Ld-ER line carryingthe ER wild type gene in Ld-er1 background (extreme right hand bar onthe figure).

FIG. 10 is a graphical representation showing carbon isotopiccomposition (per mil, y-axis) in a range of lines (numbered 1 to 19 onthe x-axis): Col-er mutants (line 1-14); the Col0 background ecotype(line 15); Ld-er1 lines (lines 16 and 17); an Ld-ER near isogenicecotype to Ld-er1 (line 18, line 3177 at NASC), and a transgenic T2Ld-ER line (line numbered 19) obtained by transformation of Ld-er1mutant with a construct carrying the wild type ER allele. The data showthat the ER allele gives less negative values indicative of increasedtranspiration efficiency.

FIG. 11 is a graphical representation showing direct measurements oftranspiration efficiency in Col-er mutants transformed with ERtransgene, under both high and low air humidity, such as occurs duringhot temperature events causing or associated with drought. Transpirationefficiency was measured by gas exchange techniques on mature leaves ofvegetative Arabidopsis rosettes, as a function of leaf-to-air vapourpressure difference (vpd) ie air humidity around the leaves. The higherthe vpd, the drier the air. Solid circles describe measurements for 5independent transgenic T2 lines homozygous for an ER transgene; theselines were generated by transforming the Col-er105 mutant (emptysquares) with a construct carrying the ER allele under control of the35S promoter. Data for null lines (ie lines that went throughtransgenesis but do not carry the ER transgene) are represented by solidsquares. This figure demonstrates complementation, across the wholerange of humidity tested, with the transpiration efficiencies in T2 ERlines being greater than those in the complemented Col-er105 mutant, andsimilar to those measured in the Col0-ER ecotype (empty triangles;background ecotype for Col-er105).

FIG. 12 is a graphical representation of an alignment of isolatedsequences with the entire coding region of the wheat ortholog of ERECTA.The position of each of the isolated sequences is shown relative to thewheat ortholog of ERECTA. Sequences are represented by either SEQ ID NO.or gene accession number.

FIG. 13 is a graphical representation of an alignment of isolatedsequences with the entire coding region of the maize ortholog of ERECTA.The position of each of the isolated sequences is shown relative to themaize ortholog of ERECTA. Sequences are represented by either SEQ ID NO.or gene accession number.

FIG. 14 is a graphical representation of a pairwise sequence alignmentof the ERECTA proteins isolated from Arabidopsis (SEQ ID NO: 2), maize(SEQ ID NO: 45), rice (SEQ ID NO: 3), Sorghum (SEQ ID NO: 5) and wheat(SEQ ID NO: 20). The alignment was performed using CLUSTALW multiplesequence alignment tool. Residues that are conserved between all speciesare indicated by asterisks (*). Conservation of the groups STA NEQK NHQKNDEQ QHRK MILV MILF HY or FYW is indicated by “:”. Conservation of thegroups CSA ATV SAG STNK STPA SGND SNDEQK NDEQHK NEQHRK FVLIM HFY isindicated by “.”. Gaps are indicated by dashes “-”.

FIG. 15 is a graphical representation of a phylogenetic tree indicatingthe relationship between each of the ERECTA proteins isolated fromArabidopsis (SEQ ID NO: 2), maize (SEQ ID NO: 45), rice (SEQ ID NO: 3),Sorghum (SEQ ID NO: 5) and wheat (SEQ ID NO: 20).

DETAILED DESCRIPTION OF THE INVENTION

Loci for Transpiration Efficiency and Their Identification

One aspect of the invention provides a locus associated with the geneticvariation in transpiration efficiency of a plant, wherein said locuscomprises a nucleotide sequence linked genetically to an ERECTA locus inthe genome of the plant.

As used herein, the term “locus” shall be taken to mean the location ofone or more genes in the genome of a plant that affects a quantitativecharacteristic of the plant, in particular the transpiration efficiencyof a plant. In the present context, a “quantitative characteristic” is aphenotype of the plant for which the phenotypic variation amongdifferent genotypes is continuous and cannot be separated into discreteclasses, irrespective of the number of genes that determine or controlthe phenotype, or the magnitude of genetic effects that single gene hasin determining the phenotype, or the magnitude of genetic effects ofinteracting genes.

By “associated with the genetic variation in transpiration efficiency ofa plant” means that a locus comprises one or more genes that areexpressed to determine or regulate the transpiration efficiency of aplant, irrespective of the actual rate of transpiration achieved by theplant under a specified environmental condition.

Preferably, the locus of the invention is linked to or comprises anERECTA allele or erecta allele, or a protein-encoding portion thereof.

As used herein, the term “ERECTA” shall be taken to refer to a wild typeallele comprising the following domains GTIGYIDPEYARTS, GAAQGLAYLHHDC,and TENLSEKXIIGYGASSTVYKC domains, wherein X means Y or H, or domainsmore than 94% identical to these domains. To the inventors' knowledge noother protein comprises these domains. Preferred ERECTA alleles comprisea nucleotide sequence having at least about 55% overall sequenceidentity to the protein-encoding region of any one of the exemplifiedERECTA alleles described herein, particularly any one of SEQ ID Nos: 1,3, 5, 7, 9, 11, 13, or 15. Preferably, the percentage identity to anyone of said SEQ ID NOs: is at least about 59-61%, or 70% or 80%, andmore preferably at least about 90%, and still more preferably at leastabout 95% or 99%.

Preferred ERECTA alleles are derived from, or present in, the genome ofa plant that is desiccation or drought intolerant, or poorly adapted forgrowth in dry or arid environments, or that suffers from reduced vigoror growth during periods of reduced rainfall or drought, or from thegenome of a plant with increased growth rate or growth duration orpartitioning of C to shoot and harvested parts under well-wateredconditions.

More preferably, an ERECTA allele is derived from, or present in, thegenome of a brassica plant, broad acre crop plant, perennial grass (eg.of the subfamily Pooidaea, or the Tribe Poeae), or tree. Even morepreferably, an ERECTA allele is present in or derived from the genome ofa plant selected from the group consisting of barley, wheat, rye,sorghum, rice, maize, Phalaris aquatica, Dactylus glomerata, Loliumperenne, Festuca arundinacea, cotton, tomato, soybean, oilseed rape,poplar, and pine.

The term “erecta” shall be taken to mean any allelic variant of thewild-type ERECTA allele that modifies transpiration efficiency of aplant

Preferred erecta alleles include the following A. thaliana erectaalleles derived from Columbia (Col) and Landsberg erecta (er) lines.Erecta alleles¹ Genomic position Lesion Affected domain Ler er-1 2249TΠA PK Col er-101 6565 TΠA PK Col er-102/106 6565 TΠA PK Col er-103 846GΠA LRR10 Col er-105 foreign DNA insert insertion Null allele between +5and +1056 Col er-108 5649 GΠA Col er-111 5749 GΠA Untranslated regionbetween LRR and transmembrane domains Col er-113 3274 C→ T Col er-1146807 G→ A PK Col er-115 3796 C→ T Col er-116 6974 G→ A PK Col er-1175203 G→ A LRR18¹alleles described by Lease et al. 2001, New Phytologist, 151: 133-143,except for Ler er-1, Col er-103 and Col-er105 which were described inTorii et al., 1996, The Plant Cell 8: 735-746

The present invention clearly encompasses an erecta allele derived from,or present in, the genome of a plant that is desiccation or droughtintolerant, or poorly adapted for growth in dry or arid environments, orthat suffers from reduced vigor or growth during periods of reducedrainfall or drought, or from the genome of a plant with increased growthrate or growth duration or partitioning of C to shoot and harvestedparts under well-watered conditions.

More preferably, an erecta allele is derived from, or present in, thegenome of a brassica plant, broad acre crop plant, perennial grass (eg.of the subfamily Pooidaea, or the Tribe Poeae), or tree. Even morepreferably, an erecta allele is present in or derived from the genome ofa plant selected from the group consisting of barley, wheat, rye,sorghum, rice, maize, Phalaris aquatica, Dactylus glomerata, Loliumperenne, Festuca arundinacea, cotton, tomato, soybean, oilseed rape,poplar, and pine.

For the purposes of nomenclature, the nucleotide sequence of theArabidopsis thaliana ERECTA protein-encoding region and the5′-untranslated region (UTR) and 3′-UTR, is provided herein as SEQ IDNO: 1. The amino acid sequence of the polypeptide encoded by SEQ ID NO:1 is set forth herein as SEQ ID NO: 2.

A particularly preferred ERECTA allele from rice (Oryza sativa) isderived from chromosome 6 of that plant species. For the purposes ofnomenclature, the protein-encoding region of the rice ERECTA gene isprovided herein as SEQ ID NO: 3. The amino acid sequence of thepolypeptide encoded by SEQ ID NO: 3 is set forth herein as SEQ ID NO: 4.

A particularly preferred ERECTA gene derived from the genome of Sorghumbicolor, is provided herein as SEQ ID NO: 5. The amino acid sequence ofthe polypeptide encoded by SEQ ID NO: 5 is set forth herein as SEQ IDNO: 6.

A further exemplary ERECTA gene derived from A. thaliana is providedherein as SEQ ID NO: 7. The amino acid sequence of the polypeptideencoded by SEQ ID NO: 7 is set forth herein as SEQ ID NO: 8.

A further exemplary ERECTA gene derived from A. thaliania is providedherein as SEQ ID NO: 9. The amino acid sequence of the polypeptideencoded by SEQ ID NO: 9 is set forth herein as SEQ ID NO: 10.

Fragments of an exemplary ERECTA gene derived from the genome of wheatare provided herein as SEQ ID NOs: 11 to 18.

An exemplary ERECTA gene derived from the genome of wheat is providedherein as SEQ ID NO: 19. The amino acid sequence of the polypeptideencoded by SEQ ID NO: 19 is set forth herein as SEQ ID NO: 20.

Fragments of an exemplary ERECTA gene derived from the genome of maizeare provided herein as SEQ ID NOs: 21 to 43.

An exemplary ERECTA gene derived from the genome of maize is providedherein as SEQ ID NO: 44. The amino acid sequence of the polypeptideencoded by SEQ ID NO: 44 is set forth herein as SEQ ID NO: 44.

The present invention clearly contemplates the presence of multiplegenes that are genetically linked or map to the specified ERECTA locuson chromosome 2. Without being bound by any theory or mode of action,such multiple linked genes may interact, such as, for example, byepistatic interaction, to determine the transpiration efficiencyphenotype.

The present invention also contemplates the presence of differentalleles of any gene that is linked to the ERECTA locus, wherein saidallele is expressed to determine the transpiration efficiency phenotype.In one embodiment, such alleles are identified by detecting a particulartranspiration efficiency phenotype that is linked to the expression ofthe particular allele. Alternatively, or in addition, the differentalleles linked to a locus are identified by detecting a structuralpolymorphism in DNA (eg. a restriction fragment length polymorphism(RFLP), amplified fragment length polymorphism (AFLP), single strandchain polymorphism (SSCP), and the like), that is linked to a particulartranspiration efficiency phenotype.

The present invention clearly encompasses all interacting genes and/oralleles that are genetically linked to an ERECTA locus and are expressedto determine a transpiration efficiency phenotype. Such linkedinteracting genes and/or alleles will map to an ERECTA locus and beassociated with the transpiration efficiency of that plant. Preferably,such interacting genes and/or alleles comprise a protein-encodingportion of a gene positioned within the ERECTA locus of the genome thatis associated with the transpiration efficiency of that plant.

Homologs and/or orthologs of the exemplified alleles are clearlyencompassed by the invention. Those skilled in the art are aware thatthe terms “homolog” and “ortholog” refer to functional equivalent units.In the present context, a homolog or ortholog of a gene that maps to anERECTA locus shall be taken to mean any gene from a plant species thatis functionally equivalent to a gene that maps to an exemplified ERECTAlocus, and comprises a protein-encoding region in its native plantgenome that shares a degree of structural identity or similarity with aprotein-encoding region of the exemplified ERECTA gene.

Preferably, a homologous or orthologous gene from a plant other than A.thaliana will be associated with the transpiration efficiency of saidplant and be linked to a protein-encoding region in its native plantgenome that comprises a nucleotide sequence having at least about 55%overall sequence identity to a protein-encoding region linked to theERECTA locus. Even more preferably, the percentage identity will be atleast about 59-61% or 70% or 80%, still more preferably at least about90%, and even still more preferably at least about 95%.

In determining whether or not two nucleotide sequences fall within aparticular percentage identity limitation recited herein, those skilledin the art will be aware that it is necessary to conduct a side-by-sidecomparison or multiple alignment of sequences. In such comparisons oralignments, differences may arise in the positioning of non-identicalresidues, depending upon the algorithm used to perform the alignment. Inthe present context, reference to a percentage identity between two ormore nucleotide sequences shall be taken to refer to the number ofidentical residues between said sequences as determined using anystandard algorithm known to those skilled in the art. For example,nucleotide sequences may be aligned and their identity calculated usingthe BESTFIT program or other appropriate program of the ComputerGenetics Group, Inc., University Research Park, Madison, Wis., UnitedStates of America (Devereaux et al, Nucl. Acids Res. 12, 387-395, 1984).In determining percentage identity of nucleotide sequences using aprogram known in the art or described herein, it is preferable thatdefault parameters are used.

Alternatively, or in addition, a homologous or orthologous ERECTA orerecta allele will be associated with the transpiration efficiency of aplant and be linked to a protein-encoding region in its native plantgenome that comprises a nucleotide sequence that encodes a polypeptidehaving at least about 55% overall sequence identity to a polypeptideencoded by a protein-encoding region linked to the ERECTA locus.Preferably, the percentage identity at the amino acid level will be atleast about 59-61% or 70% or 80%, more preferably at least about 90%,and still more preferably at least about 95%.

In determining whether or not two amino acid sequences fall within thesepercentage limits, those skilled in the art will be aware that it isnecessary to conduct a side-by-side comparison or multiple alignment ofsequences. In such comparisons or alignments, differences will arise inthe positioning of non-identical residues, depending upon the algorithmused to perform the alignment. In the present context, reference to apercentage identity or similarity between two or more amino acidsequences shall be taken to refer to the number of identical and similarresidues respectively, between said sequences as determined using anystandard algorithm known to those skilled in the art. For example, aminoacid sequence identities or similarities may be calculated using the GAPprogram and/or aligned using the PILEUP program of the Computer GeneticsGroup, Inc., University Research Park, Madison, Wis., United States ofAmerica (Devereaux et al, 1984, supra). The GAP program utilizes thealgorithm of Needleman and Wunsch, J. Mol. Biol. 48, 443-453, 1970, tomaximize the number of identical/similar residues and to minimize thenumber and length of sequence gaps in the alignment. Alternatively or inaddition, wherein more than two amino acid sequences are being compared,the ClustalW program of Thompson et al, Nucl. Acids Res. 22, 4673-4680,1994, is used. In determining percentage identity of amino acidsequences using a program known in the art or described herein, it ispreferable that default parameters are used.

Alternatively, or in addition, a homologous or orthologous ERECTA orerecta allele will be associated with the transpiration efficiency of aplant and be linked to a protein-encoding region in its native plantgenome that hybridizes to nucleic acid that comprises a sequencecomplementary to a protein-encoding region linked to an ERECTA locus,such as, for example, from A. thaliana, rice, sorghum, maize, wheat orrice. Preferably, such homologs or orthologs will be identified byhybridization under at least low stringency conditions, and morepreferably under at least moderate stringency or high stringencyhybridization conditions.

For the purposes of defining the level of stringency, a low stringencyis defined herein as being a hybridization or a wash carried out in6×SSC buffer, 0.1% (w/v) SDS at 28° C. or alternatively, as exemplifiedherein. Generally, the stringency is increased by reducing theconcentration of salt in the hybridization or wash buffer, such as, forexample, by reducing the concentration of SSC. Alternatively, or inaddition, the stringency is increased, by increasing the concentrationof detergent (eg. SDS). Alternatively, or in addition, the stringency isincreased, by increasing the temperature of the hybridization or wash.For example, a moderate stringency can be performed using 0.2×SSC to2×SSC buffer, 0.1% (w/v) SDS, at a temperature of about 42° C. to about65° C. Similarly, a high stringency can be performed using 0.1×SSC to0.2×SSC buffer, 0.1% (w/v) SDS, at a temperature of at least 55° C.Conditions for performing nucleic acid hybridization reactions, andsubsequent membrane washing, are well understood by one normally skilledin the art. For the purposes of further clarification only, reference tothe parameters affecting hybridization between nucleic acid molecules isfound in Ausubel et al., In: Current Protocols in Molecular Biology,Greene/Wiley, New York USA, 1992, which is herein incorporated byreference.

A number of mapping methods for determining useful loci and estimatingtheir effects have been described (eg. Edwards et al., Genetics 116,113-125, 1987; Haley and Knott, Heredity 69, 315-324, 1992; Jiang andZeng, Genetics 140, 1111-1127, 1995; Lander and Botstein, Genetics 121,185-199, 1989; Jansen and Stam, Genetics 136, 1447-1455, 1994; Utz andMelchinger, In: Biometrics in Plant Breeding: Applications of MolecularMarkers. Proc. Ninth Meeting of the EUCARPIA Section Biometrics in PlantBreeding, 6-8 Jul. 1994, Wageningen, The Netherlands, (J. W. van Ooijenand J. Jansen, eds), pp 195-204, 1994; Zeng, Genetics 136, 1457-1468,1994). In the present context, these methods are applied to identify themajor component(s) of the total genetic variance that contribute(s) tothe variation in transpiration efficiency of a plant, such as, forexample, determined by the measurement of carbon isotope discrimination(Δ). More particularly, the segregation of known markers is used to mapand/or characterize an underlying locus associated with transpirationefficiency. The locus method involves searching for associations betweenthe segregating molecular markers and transpiration efficiency in asegregating population of plants, to identify the linkage of the markerto the locus.

To discover a marker/locus linkage, a segregating population isrequired. Experimental populations, such as, for example, an F2generation, a backcross (BC) population, recombinant inbred lines (RIL),or double haploid line (DHL), can be used as a mapping population. Bulksegregant analysis, for the rapid detection of markers at specificgenomic regions using segregating populations, is described byMichelmoore et al. Proc Natl Acad. Sci. (USA) 88, 9828-9832, 1991. Inthe case of F2 mapping populations, F2 plants are used to determinegenotype, and F2 families to determine phenotype. Recombinant inbredlines are produced by single-seed descent. Recombinant inbred lines,such as, for example, the F9 RILs of A. thaliana (eg. Lister and Dean,Plant J., 4, 745-750, 1993) will be known to those skilled in the art.Near isogenic lines (NILs) are used for fine mapping, and to determinethe effect of a particular locus on transpiration efficiency. Anadvantage of recombinant inbred lines and double haploid lines is thatthey are permanent populations, and as a consequence, provide forreplication of the contribution of a particular locus to thetranspiration efficiency phenotype.

As for statistical methods, Single Marker Analysis (Point Analysis) isused to detect a locus in the vicinity of a single genetic marker. Themean transpiration efficiencies of a population of plants segregatingfor a particular marker, are compared according to the marker class. Thedifference between two mean transpiration efficiencies provides anestimate of the phenotypic effect of substituting one allele for anotherallele at the locus. To determine whether or not the inferred phenotypiceffect is significantly different from zero, a simple statistical test,such as t-test or F-test, is used. A significant value indicates that alocus is located in the vicinity of the marker. Single point analysisdoes not require a complete molecular linkage map. The further the locusis from the marker, the less likely it is to be detected statistically,as a consequence of recombination between the marker and the gene.

In the Anova, t-test or GLM approach, the association between markergenotype and transpiration efficiency phenotype comprises:

-   -   (i) classifying progeny of a segregating population of plants by        marker genotype, such as for example, using RFLP, AFLP, SSCP, or        microsatellite analyses, thereby establishing classes of plants;    -   (ii) comparing the mean transpiration efficiencies of classes of        plants in the segregating population, using a t-test, GLM or        ANOVA; and    -   (iii) determining the significance of the differences in the        mean at (ii), wherein a significant difference indicates that        the marker is linked to the locus for transpiration efficiency.

As will be known to those skilled in the art, the difference between themeans of the classes provides an estimate of the effect of the locus indetermining the transpiration efficiency of a class.

In the regression approach, the association between marker genotype andphenotype is determined by a process comprising:

-   -   (i) assigning numeric codes to marker genotypes; and    -   (ii) determining the regression value r for transpiration        efficiency against the codes, wherein a significant value for r        indicates that the marker is linked to the locus for        transpiration efficiency, and wherein the regression slope gives        an estimate of the effect of a particular locus on transpiration        efficiency.

For QTL interval mapping, the Mapmaker algorithm developed by Lincoln etal., Constructing genetic linkage maps with MAPMAKER/EXP version 3.0: Atutorial and reference manual. Whitehead Institute for BiomedicalResearch, Cambridge, Mass., USA, 1993, can be used. The principle behindinterval mapping is to test a model for the presence of a QTL at manypositions between two mapped marker loci. This model is a fit of apresumptive QTL to transpiration efficiency, wherein the suitability ofthe fit is tested by determining the maximum likelihood that a QTL fortranspiration efficiency lies between two segregating markers. Forexample, in the case of a QTL located between two segregating markers,the 2-loci marker genotypes of segregating progeny will each containmixtures of QTL genotypes. Accordingly, it is possible to search forloci parameters that best approximate the distribution in transpirationefficiency for each marker class. Models are evaluated by computing thelikelihood of the observed distributions with and without fitting a QTLeffect. The map position of a QTL is determined as the maximumlikelihood from the distribution of likelihood values (LOD scores: ratioof likelihood that the effect occurs by linkage: likelihood that theeffect occurs by chance), calculated for each locus.

Interval mapping by regression (Haley and Knott., Heredity 69, 315-324,1992) is a simplification of the maximum likelihood method supra whereinbasic QTL analysis or regression on coded marker genotypes is performed,except that phenotypes are regressed on the probability of a QTLgenotype as determined from the linkage between transpiration efficiencyand the nearest flanking markers. In most cases, regression mappinggives estimates of QTL position and effect that are almost identical tothose given by the maximum likelihood method. The approximation deviatesonly at places where there are large gaps, or many missing genotypes.

In the composite interval mapping (CIM) method (Jansen and Stam,Genetics 136, 1447-1455, 1994; Utz and Melchinger, 1994, supra; Zeng,Genetics 136, 1457-1468, 1994), the analysis is performed in the usualway, except that the variance from other QTLs is accounted for byincluding partial regression giving more power and precision than simpleinterval mapping, because the effects of other QTls are not present asresidual variance. CIM can remove the bias that can be caused by theQTLs that are linked to the position being tested.

Publicly available software are used to map a locus for transpirationefficiency. Such software include, for example, the following:

-   -   (i) MapMaker/QTL (ftp://genome.wi.mit.edu/pub/mapmaker3/), for        analyzing F2 or backcross data using standard interval mapping;    -   (ii) MQTL, for composite interval mapping in multiple        environments or for performing simple interval mapping using        homozygous progeny (eg. double haploids, or recombinant inbred        lines);    -   (iii) PLABQTL (Utz and Melchinger, PLABlocus Version 1.0. A        computer program to map QTL, Institut für Pflanzenzüchtung,        Saatgutforschung und Populationsgenetik, Universitat Hohenheim,        70593 Stuttgart, Germany, 1995;        http://www.uni-hohenheim.de/˜ipspwww/soft.html) for composite        interval mapping and simple interval mapping of a locus in        mapping populations derived from a bi-parental cross by selfing,        or in double haploids;    -   (iv) QTL Cartographer        (http://statgen.mcsu.edu/qtlcart/cartographer.html) for        single-marker regression, interval mapping, or composite        interval mapping, using F2 or backcross populations;    -   (v) MapQTL (http://www.cpro.dlo.nl/cbw/); Qgene for performing        either single-marker regression or interval regression to map        loci; and    -   (vi) SAS for detecting a locus by identifying associations        between marker genotype and transpiration efficiency by a single        marker analysis approach such as ANOVA, t-test, GLM or REG.

In a particularly preferred embodiment, QTL cartographer or MQTL is usedto identify a locus associated with the transpiration efficiency ofplants.

Those skilled in the art will also be aware that it is possible todetect multiple interacting alleles or genes for a particular trait,such as, for example, using composite interval mapping approaches. Toachieve this end, the composite interval mapping may be repeated to lookfor additional loci. Alternatively, or in addition, two or more distinctregions of the genome can be nominated as candidate loci, and a gameterelationship matrix constructed for each candidate locus, and a 2-locusregression performed for each pair of loci, determining a best fit forthe interacting effects between the two loci or aleles at those loci,including any dominance or additive effects. The algorithm described byCarlborg et al., Genetics (2000) can be used for simultaneous mapping.In the present context, such an analysis is performed with reference tothe segregation of transpiration efficiency phenotypes in thesegregating population.

Use of the ERECTA Locus to Enhance Transpiration Efficiency of Plants

As will be known to those skilled in the art, a single locus, if presentin the genome of a plant, can have a significant influence on thephenotype of the plant. For example, Grandillo et al., Theor. Appl.Genet. 99, 978-987, 1999, showed that for tomato a selection made from atotal 28 loci determining fruit size and weight explained 20% of thetotal phenotypic variance in this trait.

Accordingly, a second aspect of the invention provides a method ofselecting a plant having enhanced transpiration efficiency, comprising:

-   -   (a) identifying a locus associated with genetic variation in        transpiration efficiency in a plant; and    -   (b) selecting a plant that comprises or expresses a gene that        maps to the locus.

By “enhanced transpiration efficiency” is meant that the plant losesless water per unit of dry matter produced, or alternatively, producesan enhanced amount of dry matter per unit of water transpired, oralternatively, fixes an increased amount of carbon per unit watertranspired, relative to a counterpart plant. By “counterpart plant” ismeant a plant having a similar or near-identical genetic background,such as, for example, a near-isogenic plant, a sibling, or parent.

In accordance with this aspect of the invention, a locus is identifiedby conventional locus mapping means, and/or by homology searching forgenes that map to the ERECTA locus on chromosome 2 of the A. thalianagenome, such as, for example, by searching for ERECTA alleles or erectaalleles from a variety of plants, such as, for example, rice, wheat,sorghum, and maize, as described herein above.

Preferably, to select a plant that comprises or expresses theappropriate gene, marker-assisted selection (MAS) is used. As will beknown to those skilled in the art, once a particular locus has beenidentified, genetic or physical markers that are linked to the locus canbe readily identified and used to confirm the presence of the locus inbreeding populations. For a locus that is flanked by two tightly-linkedmarkers that recombine only at a low frequency, the presence of theflanking markers is indicative of the presence of the locus.

For marker-assisted selection, it is preferred that the marker is agenetic marker (eg. a gene or allele), or a physical marker (eg. leafhairiness or pod shape), or a molecular marker such as, for example, arestriction fragment length polymorphism (RFLP), a restriction (RAPD),amplified fragment length polymorphism (AFLP), or a short sequencerepeat (SSR) such as a microsatellite, or SNP. It is also within thescope of the invention to utilize any hybridization probe oramplification primer comprising at least about 10 nucleotides in lengthderived from a chromosome region that is linked in the genome of a plantto an ERECTA locus, as a marker to select plants. Those skilled in theart will readily be able to determine such probes or primers based uponthe disclosure herein, particularly for those plant genomes which mayhave sufficient chromosome sequence in the region of interest in thegenome (eg. A. thaliana, rice, cotton, barley, wheat, sorghum, maize,tomato, etc).

For flanking markers that are not tightly linked, such that there is alarge recombination distance there between, the presence of theappropriate gene is assessed by identifying those plants having bothflanking markers and then selecting from those plants having an enhancedtranspiration efficiency. Naturally, the greater the distance betweentwo markers, the larger the population of plants required to identify aplant having both markers, the intervening locus and a gene within saidlocus. Those skilled in the art will readily be able to determine thepopulation size required to identify a plant having a particulartranspiration efficiency, based upon the recombination units (cM)between two markers.

Transpiration efficiency is determined by any means known to the skilledartisan. Preferably, transpiration efficiency is determined by measuringdry matter accumulation in the plant by gravimetric means, or bymeasuring water loss, or the ratio of CO₂ assimilation rate to stomatalconductance.

In a particularly preferred embodiment, the transpiration efficiency isdetermined directly, by measuring the ratio of carbon fixed (carbonassimilation rate) to water loss (transpiration rate).

In an alternative embodiment, transpiration efficiency is determinedindirectly from the carbon isotope discrimination value (Δ). Farquhar etal., Aust. J. Plant Physiol. 9,121-137, 1982, showed that carbon isotopediscrimination (Δ; a measure of the extent to which the ¹³C/¹²C ratio oforganic matter is less than that of CO₂ in the source air), is aneffective indirect measure of transpiration efficiency. Discrimination,(Δ), is approximately the isotope ratio of carbon in source CO₂ minusthat of plant organic carbon. In a particular experiment, the source CO₂is common to all genotypes. The determination of transpirationefficiency in this manner is based upon the constancy of the atmospheric¹³C: ¹²C ratio (about 1.1:98.9) and the finding that ribulosebisphosphate carboxylase (Rubisco) enzymes discriminate against the useof ¹³C. Thus, in C₃ plants ¹³CO₂ is less efficiently assimilated than¹²CO₂, and the ¹³CO₂ left behind tends to diffuse back through stomatain and out of the leaf. However, when the stomata become nearly closed,the relative back-diffusion of ¹³CO₂ is more difficult to achieve andthe relative intracellular concentrations of ¹³CO₂ increases, therebyincreasing the proportion of this isotope that is incorporated into3-phosphoglycerate, and subsequently into dry matter. As a consequence,carbon isotope discrimination (Δ) is greatest when the overall CO₂assimilation rate during photosynthesis (A) is small, and stomatalconductance (g_(w)) to water vapor is large. This relationship isrepresented by the following algorithm:Δ(%)=27−36A/(g _(w) ×C _(a))wherein C_(a) is the ambient CO₂ concentration (ie. [¹²CO₂+¹³CO₂]).Discrimination, Δ, is approximately the isotope composition of sourceCO₂ minus that of plant organic carbon.

For a C₃ plant that exhibits a value in the range of about 4.5% to about6.7% for the term 36A/(g_(w)×C_(a)), a 1% change in carbon isotopediscrimination (Δ) corresponds to a change in transpiration efficiencyin the range of about 22% to about 15%, respectively.

The negative relationship between carbon isotope discrimination (Δ) andtranspiration efficiency has been established for many C₃ plant species,including wheat (Farquhar and Richards, Aust. J. Plant Physiol. 11,539-552, 1984; Farquhar et al., Ann. Rev. Plant Physiol. 40,388-397,1989), Stylosanthes (Thumma et al., Proc. 9^(th) Aust. Agronomy Conf.,Wagga Wagga New South Wales, Australia, 1998), cotton, barley, and rice.Accordingly, a lower carbon isotope discrimination (Δ) value for a testplant relative to a counterpart plant is indicative of enhancedtranspiration efficiency.

In C₄ species, like maize, coefficients in the equation above aredifferent (Farquhar 1983, Australian Journal of Plant Physiology,10:205-226; Henderson et al., 1992, Aust. J. Plant Physiol. 19:263-285):Δ(%)=1+5A/(g _(w) ×C _(a)).

A 1% difference in Δ corresponds to about 38% difference intranspiration efficiency. The relationship between A and transpirationefficiency is positive. ¹³C preferentially accumulates in bicarbonate,the substrate for PEP carboxylation, and so discrimination against ¹³Cis least when A is small and g_(w) is large. However, as CO₂ leakineessfrom the budle sheath increases, C₄ plants behave more like C₃ plants.

Alternatively, or in addition, transpiration efficiency is determined byanother indicator, such as, for example, leaf temperature, ash content,mineral content, or specific leaf weight (dry matter per unit leafarea). For example, specific leaf weight is positively correlated withtranspiration efficiency in peanuts and other species (Virgona et al.,Aust. J. Plant Physiol., 17, 207-214, 1990; Wright et al., Crop Sci 34,92-97, 1994). Accordingly, a higher specific leaf weight or highercarbon gain rate for a test plant relative to a counterpart plant isindicative of enhanced transpiration efficiency of the test plant.

The presence of the locus can be established by hybridizing a probe orprimer that is linked to an ERECTA locus, such as, for example, a probeor primer that hybridizes to the identified chromosome 2 region of A.thaliana or the identified chromosome 6 region of rice.

Preferably, the presence of the locus is established by hybridizing aprobe or primer derived from any one or more of SEQ ID Nos: 1, 3, 5, 7,9, 11 to 19 or 21 to 44 or from a homologous gene in another plant, or acomplementary sequence to such a sequence, to genomic DNA from theplant, and detecting the hybridization using a detection means.

In one embodiment, detection of the hybridization is performedpreferably by labelling a probe with a reporter molecule capable ofproducing an identifiable signal, prior to hybridization, and thendetecting the signal after hybridization. Preferred reporter moleculesinclude radioactively-labelled nucleotide triphosphates and biotinylatedmolecules. Preferably, variants of the genes exemplified herein,including genomic equivalents, are isolated by hybridisation undermoderate stringency or more preferably, under high stringencyconditions, to the probe.

Alternatively, or in addition, hybridization may be detected using anyformat of the polymerase chain reaction (PCR), including AFLP. For PCR,two non-complementary nucleic acid primer molecules comprising at leastabout 20 nucleotides in length, and more preferably at least 30nucleotides in length are hybridized to different strands of a nucleicacid template molecule, and specific nucleic acid molecule copies of thetemplate are amplified enzymatically. Several formats of PCR aredescribed in McPherson et al., In: PCR A Practical Approach. IRL Press,Oxford University Press, Oxford, United Kingdom, 1991, which isincorporated herein by reference.

For enhancing the transpiration efficiency of a plant wherein the locusis polymorphic, such as, for example, an allele, the method supra ismodified to include the detection of the specific allele(s) linked tothe desired enhancement. According to this embodiment, there is provideda method of selecting a plant having enhanced transpiration efficiency,comprising:

-   -   (d) identifying a locus associated with genetic variation in        transpiration efficiency in a plant;    -   (e) identifying a polymorphic marker within said locus that is        linked to enhanced transpiration efficiency; and    -   (f) selecting a plant that comprises or expresses the marker.

Standard means known to the skilled artisan are used to identify amarker within the locus that is linked to enhanced transpirationefficiency. A population of plants that is segregating for thepolymorphic marker is generally used, wherein the transpirationefficiency phenotype of plants is then correlated or associated with thepresence of a particular allelic form of the marker. As exemplifiedherein, near-isogenic or recombinant inbred lines of plants are screenedto segregate alleles at the ERECTA locus and to correlate enhancedtranspiration efficiency with the presence of the ERECTA allele asopposed to an erecta allele. Alternatively, mutations are introducedinto an ERECTA allele such as, for example, by transposon mutagenesis,chemical mutagenesis or irradiation of plant material, and mutant linesof plants are established and screened to segregate alleles at theERECTA locus that are correlated with the genetic variation intranspiration efficiency.

Suitable markers include any one or more of the markers described hereinto be suitable for MAS.

Preferably, the selection of plants in accordance with these embodimentsincludes the additional step of introducing the locus or polymorphicmarker to a plant, such as, for example, by standard breeding approachesor by recombinant means. This may be carried out at the same time, orbefore, selecting the locus or polymorphic marker.

Recombinant means generally include introducing a gene constructcomprising the locus or marker into a plant cell, selecting transformedtissue and regenerating a whole plant from the transformed tissueexplant. Means for introducing recombinant DNA into plant tissue orcells include, but are not limited to, transformation using CaCl₂ andvariations thereof, in particular the method described by Hanahan(1983), direct DNA uptake into protoplasts (Krens et al, Nature 296,72-74, 1982; Paszkowski et al., EMBO J. 3, 2717-2722, 1984),PEG-mediated uptake to protoplasts (Armstrong et al., Plant Cell Rep. 9,335-339, 1990) microparticle bombardment, electroporation (Fromm et al.,Proc. Natl. Acad. Sci. (USA), 82, 5824-5828, 1985), microinjection ofDNA (Crossway et al., Mol. Gen. Genet. 202, 179-185, 1986),microparticle bombardment of tissue explants or cells (Christou et al,Plant Physiol. 87, 671 -674, 1988; Sanford, Part. Sci. Technol. 5,27-37, 1988), vacuum-infiltration of tissue with nucleic acid, or in thecase of plants, T-DNA-mediated transfer from Agrobacterium to the planttissue as described essentially by An et al., EMBO J. 4, 277-284, 1985;Herrera-Estrella et al., Herrera-Estella et al., Nature 303, 209-213,1983; Herrera-Estella et al., EMBO J. 2, 987-995, 1983; orHerrera-Estella et al., In: Plant Genetic Engineering, CambridgeUniversity Press, N.Y., pp 63-93, 1985.

For microparticle bombardment of cells, a microparticle is propelledinto a cell to produce a transformed cell. Any suitable ballistic celltransformation methodology and apparatus can be used in performing thepresent invention. Exemplary apparatus and procedures are disclosed byStomp et al. (U.S. Pat. No. 5,122,466) and Sanford and Wolf (U.S. Pat.No. 4,945,050). When using ballistic transformation procedures, the geneconstruct may incorporate a plasmid capable of replicating in the cellto be transformed.

Examples of microparticles suitable for use in such systems include 1 to5 micron gold spheres. The DNA construct may be deposited on themicroparticle by any suitable technique, such as by precipitation.

A whole plant may be regenerated from the transformed or transfectedcell, in accordance with procedures well known in the art. Plant tissuecapable of subsequent clonal propagation, whether by organogenesis orembryogenesis, may be transformed with a gene construct of the presentinvention and a whole plant regenerated therefrom.

The particular tissue chosen will vary depending on the clonalpropagation systems available for, and best suited to, the particularspecies being transformed. Exemplary tissue targets include leaf disks,pollen, embryos, cotyledons, hypocotyls, megagametophytes, callustissue, existing meristematic tissue (eg., apical meristem, axillarybuds, and root meristems), and induced meristem tissue (eg., cotyledonmeristem and hypocotyl meristem).

The term “organogenesis”, as used herein, means a process by whichshoots and roots are developed sequentially from meristematic centres.

The term “embryogenesis”, as used herein, means a process by whichshoots and roots develop together in a concerted fashion (notsequentially), whether from somatic cells or gametes.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedto give homozygous second generation (or T2) transformant, and the T2plants further propagated through classical breeding techniques.

The generated transformed organisms contemplated herein may take avariety of forms. For example, they may be chimeras of transformed cellsand non-transformed cells; clonal transformants (eg., all cellstransformed to contain the expression cassette); grafts of transformedand untransformed tissues (eg., in plants, a transformed root stockgrafted to an untransformed scion).

Alternatively, the transformed plants are produced by an in plantatransformation method using Agrobacterium tumefaciens, such as, forexample, the method described by Bechtold et al., CR Acad. Sci. (Paris,Sciences de la vie/Life Sciences) 316, 1194-1199, 1993 or Clough et al.,Plant J. 16: 735-74, 1998, wherein A. tumefaciens is applied to theoutside of the developing flower bud and the binary vector DNA is thenintroduced to the developing microspore and/or macrospore and/or thedeveloping seed, so as to produce a transformed seed. Those skilled inthe art will be aware that the selection of tissue for use in such aprocedure may vary, however it is preferable generally to use plantmaterial at the zygote formation stage for in planta transformationprocedures.

Identification of Genes for Determining the Transpiration Efficiency ofa Plant

As exemplified herein, the inventors also identified specific genes oralleles that are linked to the ERECTA locus of A. thaliana, and rice anddetermine the transpiration efficiencies of those plants. Moreparticularly, the transpiration efficiencies of near-isogenic lines,each carrying a mutation within an ERECTA locus, and a correlationbetween transpiration efficiency phenotype and ERECTA expression or genecopy number are determined, thereby providing the genetic contributionof genes or alleles at the ERECTA locus to transpiration efficiency.This analysis permits an assessment of the genetic contribution ofparticular alleles to transpiration efficiency, thereby determiningallelic variants that are linked to a particular transpirationefficiency. Thus, the elucidation of the ERECTA locus for transpirationefficiency in plants facilitates the fine mapping and determination ofallelic variants that modulate transpiration efficiency. The methodsdescribed herein can be applied to an assessment of the contribution ofspecific alleles to the transpiration efficiency phenotype for any plantspecies that is amenable to mutagenesis such as, for example, bytransposon mutagenesis, irradiation, or chemical means known to theskilled artisan for mutating plants.

Accordingly, a third aspect of the invention provides a method ofidentifying a gene that determines the transpiration efficiency of aplant comprising:

-   -   (a) identifying a locus associated with genetic variation in        transpiration efficiency in a plant;    -   (b) identifying a gene or allele that is linked to said locus,        wherein said gene or allele is a candidate gene or allele for        determining the transpiration efficiency of a plant; and    -   (c) determining the transpiration efficiencies of a panel of        plants, wherein not all members of said panel comprise or        express said gene or allele, and wherein variation in        transpiration efficiency between the members of said panel        indicates that said gene is involved in determining        transpiration efficiency.

In another embodiment, the method comprises:

-   -   (a) identifying a locus associated with genetic variation in        transpiration efficiency in a plant;    -   (b) identifying multiple alleles of a gene that is linked to        said locus, wherein said gene is a candidate gene involved for        determining the transpiration efficiency of a plant; and    -   (c) determining the transpiration efficiencies of a panel of        plants, wherein each member of said panel comprises, and        preferably expresses, at least one of said multiple alleles,        wherein variation in transpiration efficiency between the        members of said panel indicates that said gene is involved in        determining transpiration efficiency.

In the present context, the term “near isogenic plants” shall be takento mean a population of plants having identity over a substantialproportion of their genomes, notwithstanding the presence ofsufficiently few differences to permit the contribution of a distinctallele or gene to the transpiration efficiency of a plant to bedetermined by a comparison of the transpiration efficiency phenotypes ofthe population. As will be known to the skilled artisan, recombinantinbred lines, lines produced by introgression of a gene or transposonfollowed by several generations of backcrossing, or siblings, aresuitable near-isogenic lines for the present purpose.

Preferably, the identified gene or allele identified by the methoddescribed in the preceding paragraph is selected from the groupconsisting of ERECTA allele, erecta allele, and homologs of ERECTA,wherein said homologs are from plants species other than A. thaliana.

In a particularly preferred embodiment, the identified gene or allelewill comprise a nucleotide sequence selected from the group consistingof:

-   -   (d) a sequence having at least about 55% identity to a sequence        selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:        3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ        ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID        NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 SEQ ID NO:        21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,        SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ        ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 SEQ ID NO: 33, SEQ ID        NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:        38; SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42,        SEQ ID NO: 43 and SEQ ID NO: 44;    -   (e) a sequence encoding an amino acid sequence having at least        about 55% identity to an amino acid sequence selected from the        group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 20 and        SEQ ID NO: 45; and    -   (f) a sequence complementary to (a) or (b).

Preferably, the percentage identity is at least about 59-61% or 70% or80%, more preferably at least about 90%, and even more preferably atleast about 95% or 99%. In a particularly preferred embodiment, theidentified gene or allele comprises a nucleotide sequence selected fromthe group consisting of:

-   -   (a) a sequence selected from the group consisting of SEQ ID NO:        1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ        ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ-ID NO: 14, SEQ ID        NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:        19 SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,        SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ        ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 SEQ ID        NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:        37, SEQ ID NO: 38; SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41,        SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44;    -   (b) a sequence encoding an amino acid sequence selected from the        group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 20 and        SEQ ID NO: 45; and    -   (c) a sequence complementary to (a) or (b).

Enhancement of Transpiration Efficiency using Isolated Genes

The identified gene or alleles, including any homologs from a plantother than A. thaliana, such as, for example, the wild-type ERECTAallele, or a homolog thereof, is useful for the production of novelplants. Such plants are produced, for example, using recombinanttechniques, or traditional plant breeding approaches such as byintrogression.

Accordingly, a fourth aspect of the present invention provides a methodof enhancing the transpiration efficiency of a plant comprisingectopically expressing in a plant an isolated ERECTA gene or an allelicvariant thereof or the protein-encoding region thereof.

Preferably, the ERECTA gene or allelic variant comprises a nucleotidesequence that is homologous to a protein-encoding region of a gene thatis linked to the A. thaliana ERECTA locus on chromosome 2.

In a particularly preferred embodiment, the isolated gene comprises anucleotide sequence selected from the group consisting of:

-   -   (a) a sequence having at least about 55% identity to a sequence        selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:        3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ        ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID        NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 SEQ ID NO:        21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,        SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ        ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 SEQ ID NO: 33, SEQ ID        NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:        38; SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42,        SEQ ID NO: 43 and SEQ ID NO: 44;    -   (b) a sequence encoding an amino acid sequence having at least        about 55% identity to an amino acid sequence selected from the        group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 20 and        SEQ ID NO: 45; and    -   (c) a sequence complementary to (a) or (b).

Preferably, the percentage identity is at least about 59-61% or 70% or80%, more preferably at least about 90%, and even more preferably atleast about 95% or 99%.

In a particularly preferred embodiment, the isolated gene or allelecomprises a nucleotide sequence selected from the group consisting of:

-   -   (a) a sequence selected from the group consisting of SEQ ID NO:        1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ        ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID        NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:        19 SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,        SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ        ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 SEQ ID        NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:        37, SEQ ID NO: 38; SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41,        SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44;    -   (b) a sequence encoding an amino acid sequence selected from the        group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,        SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 20 and        SEQ ID NO: 45; and    -   (c) a sequence complementary to (a) or (b).

To ectopically express the isolated gene in a plant, theprotein-encoding portion of the gene is generally placed in operableconnection with a promoter sequence that is operable in the plant, whichmay be the endogenous promoter or alternatively, a heterologouspromoter, and a transcription termination sequence, which also may bethe endogenous or an heterologous sequence relative to the gene ofinterest. The promoter and protein-encoding portion and transcriptiontermination sequence are generally provided in the form of a geneconstruct, to facilitate introduction and maintenance of the gene in aplant where it is to be ectopically expressed. Numerous vectors suitablefor introducing genes into plants have been described and are readilyavailable. These may be adapted for expressing an isolated gene in aplant to enhance transpiration efficiency therein.

Reference herein to a “promoter” is to be taken in its broadest contextand includes the transcriptional regulatory sequences of a classicaleukaryotic genomic gene, including the TATA box which is required foraccurate transcription initiation, with or without a CCAAT box sequenceand additional regulatory elements (ie. upstream activating sequences,enhancers and silencers) which alter gene expression in response todevelopmental and/or external stimuli, or in a tissue-specific manner.In the present context, the term “promoter” is also used to describe asynthetic or fusion molecule, or derivative which confers, activates orenhances expression of said sense molecule in a cell. Preferredpromoters may contain additional copies of one or more specificregulatory elements, to further enhance expression and/or to alter thespatial expression and/or temporal expression of a nucleic acid moleculeto which it is operably connected. For example, copper-responsiveregulatory elements may be placed adjacent to a heterologous promotersequence driving expression of a nucleic acid molecule to confer copperinducible expression thereon.

Placing a nucleic acid molecule under the regulatory control of apromoter sequence means positioning said molecule such that expressionis controlled by the promoter sequence. A promoter is usually, but notnecessarily, positioned upstream or 5′ of the protein-encoding portionof the gene that it regulates. Furthermore, the regulatory elementscomprising a promoter are usually positioned within 2 kb of the startsite of transcription of the structural protein-encoding nucleotidesequences, or a chimeric gene comprising same. In the construction ofheterologous promoter/structural gene combinations it is generallypreferred to position the promoter at a distance from the genetranscription start site that is approximately the same as the distancebetween that promoter and the gene it controls in its natural setting,ie., the gene from which the promoter is derived. As is known in theart, some variation in this distance can be accommodated without loss ofpromoter function. Similarly, the preferred positioning of a regulatorysequence element with respect to a heterologous gene to be placed underits control is defined by the positioning of the element in its naturalsetting, ie., the genes from which it is derived. Again, as is known inthe art, some variation in this distance can also occur.

Promoters suitable for use in gene constructs of the present inventioninclude those promoters derived from the genes of viruses, yeasts,moulds, bacteria, insects, birds, mammals and plants which are capableof functioning in plant cells, including monocotyledonous ordicotyledonous plants, or tissues or organs derived from such cells. Thepromoter may regulate gene expression constitutively, or differentiallywith respect to the tissue in which expression occurs or, with respectto the developmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, or metalions, amongst others.

Examples of promoters useful in performing this embodiment include theCaMV 35S promoter, rice actin promoter, rice actin promoter linked torice actin intron (PAR-IAR) (McElroy et el. Mol and Gen Genetics,231(1), 150-160, 1991), NOS promoter, octopine synthase (OCS) promoter,Arabidopsis thaliana SSU gene promoter, napin seed-specific promoter,PcSVMV, promoters capable of inducing expression under hydric stress, asdescribed by, for example, Kasuga et al, Nature Biotechnology, 17,287-291, 1999), SCSV promoter, SCBV promoter, 35s promoter (Kay et al,Science 236, 4805, 1987) and the like. In addition to the specificpromoters identified herein, cellular promoters for so-calledhousekeeping genes, including the actin promoters, or promoters ofhistone-encoding genes, are useful.

The term “terminator” refers to a DNA sequence at the end of atranscriptional unit which signals termination of transcription.Terminators are 3′-non-translated DNA sequences containing apolyadenylation signal, that facilitate the addition of a polyadenylatesequence to the 3′-end of a primary transcript. Terminators active incells derived from viruses, yeasts, moulds, bacteria, insects, birds,mammals and plants are known and described in the literature. They areisolatable from bacteria, fungi, viruses, animals and/or plants.

Examples of terminators particularly suitable for use in the geneconstructs of the present invention include the nopaline synthase (NOS)gene terminator of Agrobacterium tumefaciens, the terminator of theCauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator fromZea mays, the Rubisco small subunit (SSU) gene terminator sequences andsubclover stunt virus (SCSV) gene sequence terminators, amongst others.

Those skilled in the art will be aware of additional promoter sequencesand terminator sequences that may be suitable for use in performing theinvention. Such sequences may readily be used without any undueexperimentation.

Preferably, the gene construct further comprises an origin ofreplication sequence for its replication in a specific cell type, forexample a bacterial cell, when said gene construct is required to bemaintained as an episomal genetic element (eg. plasmid or cosmidmolecule) in said cell. Preferred origins of replication include, butare not limited to, the fl-ori and col/E1 origins of replication.

Preferably, the gene construct further comprises a selectable markergene or genes that are functional in a cell into which said geneconstruct is introduced.

As used herein, the term “selectable marker gene” includes any genewhich confers a phenotype on a cell in which it is expressed tofacilitate the identification and/or selection of cells which aretransfected or transformed with a gene construct of the invention or aderivative thereof.

Suitable selectable marker genes contemplated herein include theampicillin resistance (Amp^(r)), tetracyclin-resistance gene (Tc^(r)),bacterial kanamycin resistance gene (Kan^(r)), phosphinothricinresistance gene, neomycin phosphotransferase gene (nptII), hygromycinresistance gene, gentamycin resistance gene (gent), β-glucuronidase(GUS) gene, chloramphenicol acetyltransferase (CAT) gene, and luciferasegene, Green Fluorescent Protein gene (EGFP and variants), amongstothers.

In a related embodiment, the invention extends to the use of an isolatedgene comprising a nucleotide sequence that is homologous to aprotein-encoding region of a gene of A. thaliana that is positionedbetween about 46 cM to about 50.74 cM on chromosome 2 in the preparationof a gene construct for enhancing the transpiration efficiency of aplant.

In an alternative embodiment of the invention, the transpirationefficiency of a plant is enhanced by classical breeding approaches,comprising introgressing the isolated gene into a plant. Forintrogression of a gene, the gene is transferred from its native geneticbackground into another genetic background using standard breeding, forexample, a gene that enhances transpiration efficiency in a progenitorsuch as a diploid cotton or diploid wheat may be transferred into acommercial tetraploid cotton or hexaploid wheat, respectively, bystandard crossing, followed by several generations of back-crossing toremove the genetic background of the progenitor. Naturally, continuedselection of the gene of interest is required, such as, for example,facilitated by the use of markers.

A further aspect of the present invention provides a plant havingenhanced transpiration efficiency, wherein said plant is produced by amethod described herein.

Clearly the ERECTA genes, allelic variants and protein coding regionsdescribed herein are useful in determining other proteins that areinvolved in the transpiration process in plants. For example, an ERECTAgene, allelic variant thereof or protein coding region thereof may beused in a forward ‘n’-hybrid assay to determine if said peptide is ableto bind to a protein or peptide of interest. Forward ‘n’ hybrid methodsare well known in the art, and are described for example, by Vidal andLegrain Nucl. Acid Res. 27(4), 919-929 (1999) and references therein,and include yeast two-hybrid, bacterial two-hybrid, mammaliantwo-hybrid, PolIII (two) hybrid, the Tribrid system, the ubiquitin basedsplit protein sensor system and the SOS recruitment system. Such methodsare incorporated herein by reference

In adapting a standard forward two-hybrid assay to the presentinvention, an ERECTA protein is expressed as a fusion protein with a DNAbinding domain from, for example, the yeast GAL4 protein. Methods ofconstructing expression constructs for the expression of such fusionproteins are well known in the art, and are described, for example, inSambrook et al (In: Molecular Cloning: A laboratory Manual, Cold SpringHarbour, New York, Second Edition, 1989). A second fusion protein isalso expressed in the yeast all, said fusion protein comprising, forexample, a protein thought to interact with an ERECTA protein, forexample the GAL4 activation domain. These two constructs are thenexpressed in a yeast cell in which, a reporter molecule (e.g., tet^(r),Amp^(r), Rif, bscdf, zeof, Kan^(r), gfp, cobA, LacZ, TRP1, LYS2, HIS3,HIS5, LEU2, URA3, ADE2, MET13, MET15) under the control of a minimalpromoter placed in operable connection with a GAL 4 binding site. If theproteins do not interact, a reporter molecule is not expressed. However,if said proteins do interact, said reporter molecule is expressed.Accordingly a protein, polypeptide, peptide that is able to specificallybind a target protein is identified.

A forward ‘n’-hybrid method may be modified to facilitate highthroughput screening of a library of peptides, polypeptides and/orproteins in order to determine those that interact with an ERECTAprotein. Methods of screening libraries of proteins are well known inthe art and are described, for example, in Scopes (In: ProteinPurification: Principles and Practice, Third Edition, Springer Verlag,1994). Proteins identified by this method are potentially involved inthe transpiration process in plants.

The present invention is further described with reference to thefollowing non-limiting examples.

EXAMPLE 1 ¹²C/¹³C Discrimination as a Marker for Screening GeneticVariation in Transpiration Efficiency

Experimental conditions and sampling procedures were established toallow the control of many factors, other than genetic, that influencetranspiration efficiency at the level of individual leaves and plants.These factors fall into several categories: (a) characteristics of theseedling's micro-environment: temperature, light, humidity, boundarylayer around the leaves, root growth conditions; (b) developmental andmorphological effects that modify gas exchange and C metabolism andtherefore carbon isotopic signature (eg age, stage, posture); and (c)seed effects.

We developed high resolution mass-spectrometer techniques for measuringC isotope ratios in whole tissues or carbon compounds such as solublesugars—ie a measure of integrated transpiration efficiency over theplant's life or over a day, respectively, and also for measuringinstantaneous transpiration efficiency during gas exchange.

This means:

-   -   0.1 per mil analytical precision in the measurement of the        isotopic composition of leaf carbon. Discrimination, (Δ), is        approximately the isotope ratio of carbon in source CO₂ minus        that of plant organic carbon. In a particular experiment, the        source CO₂ is common to all genotypes.    -   0.1 per mil biological precision, that is variation between        replicated seedlings, grown in soil, either in growth chambers        or in glasshouses with CO₂, humidity and temperature control        (corresponding to approximately 1.5% variation in transpiration        efficiency).    -   The ability to grow and screen large batches of seedlings in        glasshouses or growth chambers (up to 1500), under standardised        leaf and root growth conditions, to a rosette size of several cm        within 2-3 weeks allowing individual measurements, on the same        plant, of isotope ratios and also of the underlying properties        (eg in situ measurement of leaf temperature by infra-red        thermometry as a measure of stomatal conductance; chlorophyll        fluorescence; leaf expansion).

EXAMPLE 2 Natural Genetic Variation in Transpiration Efficiency inArabidopsis thaliana

A. thaliana ecotypes were screened for leaf Δ under glasshouseconditions. There was a large spread of values (corresponding toapproximately 30% genetic variation in transpiration efficiency).However, large environmental effects were noted. A few contrastedecotypes were selected at the two extremes of the range of Δ values andcompared under various conditions of irradiance (150 to 500 μE m⁻²s⁻¹),light spectrum (Red/Far-Red ratios) and air humidity (60 to 90%) whileroots were always well watered. The magnitude of genetic differences intranspiration efficiency was very much influenced by environmentalconditions. This was in part due to variations among ecotypes in thedependence of photosynthesis on light and vapour pressure deficit.Genetic differences were maximal under a combination of high light andlow humidity, in growth chambers.

The ecotypes Columbia (Col) and Landsberg erecta (Ld-er) have extremecarbon isotope discrimination values, with Col always having smaller Δvalues than Ld-er ie less negative δ¹³C isotopic compositions, and thusa greater transpiration efficiency.

EXAMPLE 3 Identification of a Locus Associated with TranspirationEfficiency in A. thaliana

Quantitative Trait Loci (QTL) analysis of the Lister and Dean's (1993)Recombinant Inbred Lines (later referred to as RILs) was performed toidentify and map a locus associated with carbon isotope discrimination(Δ). The RILs were from a cross between Col-4 and Ler-0. Our analysisshowed the importance of genes around the ER locus on chr2, and a rolefor genes other than ERECTA in conferring transpiration efficiency on A.thaliana.

More particularly, 300 RI mapping lines between Col and Ler ecotypes,available at the Arabidopsis Stock Centre, were generated from a crossbetween the Arahidopsis ecotypes Columbia (Col4) and Landsberg erecta(Ler-0 carrying er1) (Lister and Dean, 1993), using Columbia as the maleparent. A subset of 100 of these lines, chosen as the most densely andreliably mapped were used in the present analysis.

The seeds were multiplied in a glasshouse in an attempt to minimizeconfounding seed effects in our comparisons. Large numbers of seeds wereobtained for most lines except for a few, including Col4 parent, whichhad to be re-ordered following low seed viability of the original samplesent by the Stock Centre. The seeds harvested in these propagation runswere used throughout all our experiments to date.

Loci were analysed using two programs, QTL cartographer and MQTL. Theseprograms compute statistics of a trait at each marker position, using arange of methods [linear regression (LR), stepwise regression (SR), andlikelihood approaches (Single interval mapping (SIM) which treats valuesat individual markers as independent values, and composite intervalmapping (CIM) which allows for interactions between markers andassociated locus)]. By nature each of these methods has some biases andembedded assumptions, hence the importance of analysing data with morethan one program. Only results that were consistent between the twoprograms, and robust to additions or deletions to the set of backgroundmarkers used for composite interval mapping are reported below.

Initial QTL analysis was done in parallel to seed multiplication on asubset of 40 lines for which enough seeds were sent. Once all seeds hadbeen multiplied this was repeated on the full set of 100 lines. Thesetwo analyses indicated the existence of a locus for carbon isotopediscrimination (Δ), that maps to the region including the ERECTA locuson chromosome 2, at approximately 46-51 cM (Table 1, run 1&2).

Given the complexity and integrative nature of Δ as a physiologicaltrait, such a small number of loci associated with the trait was notexpected. Subsequent experiments were therefore designed to test theseresults and assess their stability across the range of environmentalconditions known for their effects on gene expression related to Δ (seeabove). QTL analysis was repeated on several completely independent datasets obtained under highly controlled conditions in glasshouses orgrowth chambers, where either air humidity, photoperiod or irradiance(amount, diurnal pattern, day to day variation) was varied. Depending onthe experiment, all 100 recombinants inbred lines were included or onlythe subset of lines with cross-overs on chromosome 2. These experimentsconfirmed that genetic variation in Δ could be mostly ascribed to aportion of chromosome 2 (Table 1) between about 46-50.7 cM.

When RILs were sorted graphically according to carbon isotopediscrimination and their genotype at the ER marker (50.64 cM) and itsvicinity (Ld-er1 genotype or Col-ER genotype), lines which were Ld-er atthe ERECTA marker ranked mostly at the high end of carbon isotopediscrimination values. In contrast, lines having a Col-ERECTA markergenotype ranked mostly at the low end of carbon isotope discriminationvalues (data available on request). In the middle of the range of carbonisotope discrimination values, there was some overlap between the twosets of lines. Some lines were always at an extreme (in all 18experiments performed), while the ranking of other lines was moreunstable. These data indicate a locus for transpiration efficiency, asdetermined by the carbon isotope discrimination value, in the vicinityof the ERECTA locus on chromosome 2 (Table 1). This locus most likelyinvolves the ER gene. Depending on the positions of cross-overs betweenLd-er and Col, recombination between ERECTA and one or more of the othergenes influences the transpiration efficiency phenotype of the progeny.

EXAMPLE 4 Transformation Protocol for Maize

Gun Transformation

A suitable method for maize transformation is based on the use of aparticle gun identical to that described by J. Finer (1992, Plant CellReport, 11:323-328). The target cells are fast dividing undifferentiatedcells having maintained a capacity to regenerate in whole plants. Thistype of cells composes the embryogenic callus (called type II) of maize.These calluses are obtained from immature embryos of genotype Hillaccording to the method and on medium described by Armstrong (MaizeHandbook; 1994 M. Freeling, V. Walbot Eds ; pp.665-671).

These fragments of the calluses having a surface from 10 to 20 mm2 arearranged, 4 hour before bombardment, by putting 16 fragments by dish inthe center of a Petri dish containing an culture medium identical to themedium of initiation of calluses, supplemented with 0.2 M of mannitol+0.2 M of sorbitol. Plasmids containing the ERECTA sequences to beintroduced, are purified on QiagenR column following the instructions ofthe manufacturer.

They are then precipitated on particles of tungsten (M10) following theprotocol described by Klein et al. Nature, 327, 70-73, (1987). Particlesso coated are sent towards the target cells by means of the gunaccording to the protocol described by Finer et al. Plant Cell Report,11:323-328, 1992. The bombarded dishes of calluses are then sealed bymeans of ScellofraisR then cultivated in the dark at 27° C.

The first transplanting takes place 24 hours later, then every otherweek during 3 months on medium identical to the medium of initiationsupplemented with a selective agent. After 3 months or sometimesearlier, one can obtain calluses the growth of which is not inhibited bythe selective agent, usually and mainly consisting of cells resultingfrom the division of a cell having integrated into its genetic patrimonyone or several copies of the gene of selection. The frequency ofobtaining of such calluses is about 0.8 callus by bombarded dish.

These calluses are identified, individualized, amplified then cultivatedso as to regenerate seedlings, by modifying the hormonal and osmoticequilibrium of the cells according to the method described by Vain andal. (1989, Plant Cell tissue and organ Culture 18:143-151). These plantsare then acclimatized in greenhouse where they can be crossed forobtaining hybrids or self-fertilized.

In a preferential way, one can use a similar protocol, the principle ofwhich is described in Methods of Molecular Biology: Plant gene transferand expression protocols (1995, vol. 49, PP113-123), and in which theimmature embryos of genotype Hill are directly bombarded with goldenparticles coated with plasmides ERECTA to introduce, prepared accordingto the protocol described by Barcelo and Lazzeri (1995, Methods ofMolecular Biology, 49:113-123).

Steps of transformation, selection of the events, maturation andregeneration are similar to those described in the previous protocol.

Agrobacterium Transformation

Another technique of useful transformation within the framework of theinvention uses Agrobacterium tumefaciens, according to the protocoldescribed by Ishida and al (1996, Nature Biotechnology 14: 754-750), inparticular starting from immature embryos taken 10 days afterfertilization.

All the used media are referenced in the quoted reference. Thetransformation begins with a phase of co-culture where the immatureembryos of the maize plants are put in contact during at least 5 minuteswith Agrobacterium tumefaciens LBA 4404 containing the superbinaryvectors.

The superbinary plasmid is the result of an homologous recombinationbetween an intermediate vector carrying the T-DNA, and containing thegene of interest and/or the marker gene of selection, and the vectorpSB1 of Japan Tobacco (EP 672 752) containing: the virB and virG genesof the plasmide pTiBo542 present in the supervirulent strain A281 ofAgrobacterium tumefaciens (ATCC 37349) and an homologous region found inthe intermediate vector, allowing homologous recombination.

Embryos are then placed on LSAs medium for 3 days in the dark and at 25°C. A first selection is made on the transformed calluses: embryogeniccalluses are transferred on LSD5 medium containing phosphinotricine (5mg/l) and céfotaxime (250 mg /l) (elimination or limitation ofcontamination by Agrobacterium tumefaciens).

This step is performed during 2 weeks in the dark and at 25° C. Thesecond step of selection is realized by transfer of the embryos whichdeveloped on LSD5 medium, on LSD10 medium (phosphinotricine, 10 mg/l) inthe presence of cefotaxime, during 3 weeks at the same conditions aspreviously. The third stage of selection consists in excising thecalluses of type I (fragments from 1 to 2 mm) and in transferring themfor 3 weeks in the darkness and at 25° C. on LSD 10 medium in thepresence of céfotaxime. The regeneration of seedlings is made byexcising the calluses of type I which proliferated and by transferringthem on LSZ medium in the presence of phosphinotricine (5 mg/l) and ofcefotaxime for 2 weeks at 22° C. and under continuous light.

Seedlings having regenerated are transferred on RM medium+G2 containingAugmentin (100 mg/l) for 2 weeks at 22° C. and under continuousillumination for the development step. The obtained plants are thentransferred to the phytotron with the aim of acclimatizing.

EXAMPLE 5 Detecting Expression of ERECTA Protein

Extraction of ERECTA from Leaves and Seeds of Maize.

Leaves are harvested and immediately frozen in liquid nitrogen. Grindingis made in a mortar cleaned in ethanol 100% and cooled on ice. A foliardisc of 18 mm diameter is extracted in 200 μL of extraction buffer:Tris-HCl pH 8.0, glycerol 20%, MgC2 10 mM, EDTA 1 mM, DTT 1 mM, PVPinsoluble 2% (p/v), Fontainebleau sand et protease inhibitors: leupeptin2 mg/L, chymostatin 2 mg/L, PMSF 1 mM and E64 1 mg/L. The groundmaterial is then centrifuged in 4° C. during 15 minutes at 20000 g toeliminate fragments.

Grains are first reduced to powder in a bead-crusher (Retsch). Proteinsare extracted by suspending 100 μL of powder in 400 μL of the previouslydescribed buffer on ice. This mixture is vortexed and centrifuged at 4°C. during 15 minutes et 20000 g to eliminate fragments.

ERECTA protein levels are then measured using techniques known to thoseskilled in the art, and described, for example, in Scopes (In: Proteinpurification: principles and practice, Third Edition, Springer Verlag,1994).

EXAMPLE 6 Determination of a Role for the ERECTA Gene in RegulatingTranspiration Efficiency

We compared Col and Ler ecotypes with near-isogenic mutant lines for theerecta gene, to examine a possible role of the ERECTA gene indetermining carbon isotope discrimination (Δ).

Plants expressing the wild type ERECTA gene (SEQ ID NO: 1), or an erectamutant allele in the Columbia background (eg. Col-er1, Col-er2,Col-er101 to -er105; or Col-er106 to -er123) and in Landsberg background(Ld-er1) have been publicly described.

Three of these mutants were available for comparison to the isogenic ornear-isogenic lines (Table 2).

Col4 (ER) and Ld-er1, the parental lines for Lister and Dean's RILs weresystematically included in the comparison. Where possible, other Col“ecotypes” were also included, (eg. Col0, Col1, Col3-7), to assess theirsimilarity with respect to carbon isotope discrimination, especiallycompared to the RIL parental ecotype Col4.

The results of these comparisons are described in Table 3. Data indicatethe differences in carbon isotope discrimination values between er andER lines for 15 different experimental runs corresponding to growthunder low to high light (100 to 800 μE m⁻² s⁻¹), low to high humidity(40 to 85%), short to long days (8, 10, 24 hrs), normal to hightemperatures (22/20° C. to 28/20° C.).

As expected, the spread of carbon isotope discrimination values amonglines varied with environmental conditions. Lines carrying er mutationshave a greater carbon isotope discrimination value overall than thosehaving the ER wild type gene (see Table 3, column 1), indicative of alower water use-efficiency. There is usually little difference in Cisotopic discrimination between the various Col lines, (see the similaraverages obtained for columns 2, 3, and 4 in Table 3, wherein er105 iscompared to 3 different Col ecotypes, Col0, Col4 and 3176 or Col1). Whenpresent, the er105 mutant always has the greatest carbon isotopediscrimination value of all lines, including er1 and er2 (columns 2-4compared to columns 5-6 in Table 3, or column 8 compared to column 9 inTable 3). The value measured in the er105 mutant is always significantlygreater than in the ER isogenic line (column 4 in Table 3). The valuemeasured in er1 (Landsberg parental line NW20) is usually also greaterthan that in the ER lines 3177 (near isogenic, column 6 of Table 3), andto a lesser extent Col4 (Columbia parental line, column 7 of Table 3).These observations give direct evidence that the ERECTA gene plays asignificant role in determining genetic differences in carbon isotopicdiscrimination in Arabiclopsis.

This conclusion is independently confirmed by leaf gas exchangemeasurements that allow the direct measure of transpiration efficiency(ratio of net CO₂ fixation to water loss; column 4 in Table 4; FIGS. 1a-1 c, 2 a-2 c). Measurements on mature leaves reveal that ER lines arecharacterised by a greater ratio of CO₂ assimilation to water loss thanlines carrying er mutations. This is most obvious when comparing thepair Col1/er105 with a 21% greater transpiration efficiency (ratio A/E)in Col1 than er105, or the pair Col1/er2 with a 16% greatertranspiration efficiency in Col1. Consistent with the measurements ofcarbon isotope discrimination, the effect er/ER is relatively smaller inthe Ld background (9% greater ratio A/E in Ld-ER (3177) than in theLd-er1 (NSW20) line).

Also consistent with the carbon discrimination measurements, is the 20%difference in transpiration efficiency between the two RILs parentallines (4.06 and 3.38 mmolC/molH2O in Col4-ER and Ld-er1, respectively).

The fact that of all 3 erecta mutants examined, er105 has the mostextreme carbon discrimination and transpiration efficiency phenotypessuggests that the er105 mutation affects a more crucial part of theERECTA gene than er2 or er1. This is consistent with the published dataon the er105 mutant. This mutation corresponds to the insertion of alarge “foreign insert” in the ERECTA gene. The insertion inhibitstranscription of the gene and causes the strongest erecta phenotype ofall erecta mutants isolated in Col (with respect to inflorescenceclustering and silique width and shape). Alternatively, or in addition,data indicate that erecta mutations have a stronger effect on carbonisotope discrimination values in a Columbia genetic background than in aLandsberg background (comparison of phenotypic effects of er105 ander1), implying that other genes, polymorphic between Landsberg andColumbia ecotypes, interact with ERECTA in determining transpirationefficiency. This could also account for the greater difference intranspiration efficiency between er/ER lines in Col background than in aLd background (see above, Table 4). Alternatively, or in addition, dataindicate that the erecta mutation is not the only mutation present inthe er105 mutant. For example, the mutagenized Col seeds may havecarried the gl1 mutation, induced by the fast neutron irradiation, thatalso contributes to the phenotype observed.

A comparison of transcript profiles in er/ER isogenic lines (in both Coland Ld background) allows determination of the involvement of additionalgenes to ERECTA and the effect of environment on their expression.

EXAMPLE 7 QTL Detection Centred on the ERECTA Marker and ERECTA GeneLocus on Chromosome 2 of Arabidopsis thaliana

1. Methods

Numerous runs using the Lister and Dean (1993) Recombinant Inbred Linesbetween Col-4 and Ler-0 were grown in a temperature controlled glasshouse (20/20° C.) or within growth cabinets (21° C. and light levelsranging from 100 to 500 μE m⁻² s⁻¹ irradiance, and 50-70% relativehumidity). Runs included a variety of all 100 RILs as well as subsets ofthese 100 along with parental Col-4 and Ler-0 (NW20) parental lines.Individual RILs were replicated within runs. Seeds were eithercold-treated on moist filter paper for 2-4 days, cold-treated andplanted directly onto soil; or plated onto agar, cold-treated for 2-4days, grown on agar for about 11-15 days before being transferred tosoil. Plants were well-watered, and grown for 4-5 weeks before harvest.Samples (whole or part of rosette) were collected and dried in an 80° C.oven before being ground and analysed for C isotopic composition. Thevalue used for QTL analysis for an individual line was the average ofthe replicated plants of that line within one run.

2. Marker Selection

The standard set of 64 markers for the Lister and Dean recombinant lineswere down-loaded from the NASC website. Additional markers were added tothis data set when significance was first determined to get finer scalemapping in the regions of interest. A total of 121 markers were usedacross the 5 chromosomes.

3. Analysis

Runs were analysed using Simple Interval Mapping (SIM)(Lander & Botstein1989) and Composite Interval Mapping (CIM) (Zeng 1993 & 1994). Twoprograms were used to analyses the data, QTL Cartographer version 1.14(Basten et al. 1999) and MQTL version 0.98 (Tinker and Mathers 1995).The two programs differ in how they deal with background markers forComposite Interval Mapping (CIM). Within MQTL the background markers arechosen at random and put into the map input file. Within QTLCartographer the background markers are not chosen at random but ratherare chosen from the Stepwise Regression analysis selecting the “best”background markers. The setting or choosing of these markers also has aninfluence on the level of statistical significance. Tinker argues thatit is not possible to find an appropriate threshold for statisticalerror control when background markers are selected based on the data.Hence we used the two programs and have concentrated on QTLs that werepresent in both sets of analysis.

a) QTL Cartographer

Qstat was used to determine whether the data had a normal distribution(if not then measures were taken to fix the distribution). LinearRegression (LR) and Stepwise Regression (SR) were performed using thedefault settings (Stepwise regression used forward with backwardelimination) 5% significance. Simple Interval and CIM were performedusing the Zmap.qtl function. The data were analysed across allchromosomes with a walking speed of 2 cM. For model 6 (CIM) the numberof background parameters was left at the default of 5 along with thewindow size which was left on the default of 10 cM. One thousandpermutations were performed within CIM (Churchill and Doerge 1994). Eqtlwas then run to determine the significant QTLs.

b) MQTL

The same set of markers used in QTL Cartographer was used in MQTL.Background markers were chosen at random for CIM. The number of markerschosen was approximately half that of the number of RILs used in theset. The default setting of a walking speed of 5 cM was selected, 3000permutations were performed to determine significance levels with type 1error set at 5%.

QTLs that were present in both programs and from varied backgroundmarker sets from within MQTL were considered genuine. This, coupled withrepeated QTL analysis across independent experiments, lead to asignificant repeatable locus surrounding the ERECTA gene on Chromosome 2(Table 5).

Data in Table 5 indicate that there is a major QTL with a LOD scoresignificant at the probability level of 5% and, for most runs, of even1%, on Arabidopsis thaliana chromosome 2. In all cases, that intervalsits above the ER marker on chromosome 2. Depending on the experimentalrun, this QTL explains 18 to 64% (see column R²) of the total geneticvariance in transpiration efficiency.

Data in FIG. 3 indicate a positive additive effect of the identified QTLbased upon the mean value of the carbon isotope composition in plantscarrying the Col-4 ERECTA allele.

EXAMPLE 8 Complementation Test: Transformation of A. thaliana LinesCarrying Erecta Mutations with the Wild Type ERECTA Gene Under theControl of the 35S Promoter

1. Methods

Two Columbia erecta lines were transformed using a binary vectorgenerously given by Dr Keiko Torii. That plasmid was constructed usingthe vector plasmid pPZP222 (see details on this vector in Hajdukiewicset al. Plant Mol Biol 25, 989-994, 1994).

The pPZP vectors carry chimeric genes in a CaMV 35S expression cassettethat confer resistance to kanamycin or gentamycin in plants. The plantselectable marker (gentamycin resistance gene for the pPZP222 vector) iscloned next to the LB. Cloning sites for the gene of interest (ER in ourcase) is between the plant marker and the RB sequences. This ensuresthat that gene is transferred to plant first, followed by the gent gene.Resistance to gentamycin will therefore be obtained only if the ER geneis also present.

The binary vector was transferred to disarmed strain AGL1 ofAgrobacterium tumefaciens by standard tri-parental matings (Ditta et al,1980, PNAS 77,7347-7351) using the pRK2013 helper strain of E coli.

Arabidopsis plants were transformed using the standard floral dip methodfor transformation by disarmed strains of A tumefaciens (Clough andBent, 1998, The Plant Journal 16, 735-743).

Two Columbia erecta lines were transformed, for which we had numerousdata showing consistently more negative isotopic values in those lines(ie lower transpiration efficiency) than in near-isogenic Col ER wildtype plants. These two lines were as follows:

-   -   1. er 105, a knock-out mutant due to the insertion of a large        piece of DNA in the ERECTA gene and    -   2. line Col-er2 (3401 NASC identifier), same as er106 (Lease et        al.2001).

Seedlings were screened on MS plates on 100 μg/ml gentamycin sulfate.Putative transformants were transferred to soil and their progenyscreened again for gentamycin resistance, for confirmation andidentification of homozygous lines and T3 seed collection.

Many independent transformant lines were obtained and among those wereseveral ER homozygous lines, which were selected for subsequent analysis(see Table 6).

A stable transgenic homozygous Landsberg ER line also obtained bytransforming the Ld-er1 ecotype (NW20) with the same construct asdescribed above was given to us by Dr Keiko Torii (line T3-7K in Table 6or “T2+ER” in FIGS. 6-9).

2. Results:

Initial analysis of several ER transformants in the Col-er105,Col-er106/er2, and Ld-er1 background (as shown in Table 6 above):

Effective transformation was ascertained and ER expression levels werequantified in several independent T2 transformants using real-timequantitative PCR (ABI PRISM 7700, Sequence Detection System UserBulletin #2. 1997). Basically that technique allowed us to quantify thecopy number of the ER gene in lines of interest, after normalisation tothe copy number of a control gene, in the same plants (same RNA pool).18S ribosomal RNA gene was used as a control gene after checking itsexpression was not affected by changes in ER expression.

Results are shown in FIGS. 4 a, 4 b and 4 c, wherein the y-axis in eachfigure describes the erecta mRNA copy number (normalised to that of 18SmRNA) in wild type ER lines, er mutants, and ER transgenics in bothColumbia and Landsberg backgrounds.

All ER transgenic lines, except line 145 (FIG. 4 a) showed increasedmRNA copy number: from 4 to 170 fold increase compared with the nullcontrols. Interestingly, all lines, even those with hugely increasedmRNA levels look “normal”, healthy and of similar size.

Initial phenotypic analysis shows complementation of the “transpirationefficiency phenotype”. In other words, ER transgenic lines show lessnegative carbon isotopic composition values than null er control andnull lines as shown in Table 7. Those values converge towards valuesmeasured for wild type ER ecotypes. Hence in a Columbia background, ERtransgenics display values of −30.6 to −31.2 per mil on average comparedto values of −31.7 to −32.2 per mil in the null transgenics (Table 7),and −30.9 per mil in the Col0 ER wild type (background ecotype formutant er-105). The less negative carbon isotopic compositions in ERtransgenics is indicative of greater transpiration efficiency in theseplants, as expected.

The data presented in Table 7 are confirmed by direct measurement ofleaf transpiration efficiency (ratio A/E of CO₂ assimilation rate perunit leaf area to transpiration rate) using gas exchange techniques.Stomatal density, leaf photosynthetic capacity and growth rate are alsodetermined to analyze the underlying causes of the reversion of thetranspiration efficiency phenotype (leaf development and anatomy,biochemical properties of leaves, stomatal characteristics).

EXAMPLE 9 Tissue Specificity in the Expression of the ERECTA Gene inWild Type Rice Oryza sativa (cv Nipponbare)

An ortholog of the A. thaliana ERECTA allele (SEQ ID NO: 1) in rice wasidentified in silico by homology searching of the NCBI protein databaseusing the BLAST programme under standard conditions. The input sequencewas SEQ ID NO: 2. The nucleotide sequence of the rice ortholog ispresented in SEQ ID NO: 3, with the encoded protein comprising the aminoacid sequence set forth in SEQ ID NO: 4.

The mRNA copy number of the rice ERECTA gene was determined for variousplant organs/parts, as indicated in FIGS. 5 a and 5 b. ERECTA mRNA copynumbers were determined by quantitative real-time PCR, using 18S mRNA asan internal control gene for normalization of data. The pattern ofERECTA expression in rice was similar to the pattern of gene expressionin A. thaliana, with highest expression observed in young meristematictissues, young leaves and even more, the inflorescences. No or very lowexpression is found in roots, as for A. thaliana.

These similarities in tissue specificity between rice and Arabidopsisindicates that the rice orthologue provided herein as SEQ ID NO: 3 is atrue orthologue of the A. thaliana ERECTA allele set forth in SEQ ID NO:1, with similar function

EXAMPLE 10 Demonstration of a Functional Role for the Rice ERECTA Genein Modulating Transpiration Efficiency

To determine a functional role for the rice ERECTA gene (SEQ ID NO: 3),lines of rice plants carrying transposon insertions that affectexpression of that gene are analyzed.

Nine such mutants were identified in the publicly available collectionof transposon TOS17 insertional mutants at the Japanese NIAS Institute.The TOS17 retrotransposon is described in detail by Hirochika, CurrentOpinion in Plant Biology, 4, 118-122, 2001 and by Hirochika Plant MolBiol 35, 231-240, 1997, which is incorporated herein by reference. Thenine mutant lines were identified through the website URLhttp://tos.nias.affrc.go.jp/˜miyao/pub/tos17/, and they have theaccession numbers NG0578 (mutant A), ND3052 (mutant B), ND4028 (mutantC), NC0661 (mutant D), NE1049 (mutant E), NF8517 (mutant F), NE8025(mutant G), NE3033 (mutant H) and NF8002 (mutant I).

Nine transposon insertional mutants were ordered from NIAS, that carrythe TOS17 stable retrotransposon insert in various parts of the ERECTAgene in the Nipponbare background, the genotype used for rice genomesequencing: NG0578, ND3052, ND4028, NC0661, NE1049, NF8517, NE8025,NE3033 and NF8002.

The transposon insertions in these nine lines affect the membranespanning region of the protein (mutants I, D, E) or the Leucine RichRepeat (LRR) domains (mutant H and G) in LRR 7 and LRR 18, respectively.In mutant B, TOS17 alters the coding sequence just upstream of sequencesencoding the protein kinase domain I. In mutants C and F, the TOS17insertion alters the sequence encoding domain VIa of the ERECTA protein.In mutant A, the TOS7 insertion is in a sequence encoding a regionbetween domains IX and X. The sequence information on these mutants ispublicly available from the NTAS website.

Using mutant seed for lines A-I received from NIAS, plants were grownfor amplification of seed and analysis. Except in two mutants whereseveral plants died, plants look healthy, with good growth indicatingthat, as in Arabidopsis, there is great potential to alter the ERECTAgene towards altered transpiration efficiency without adverselyaffecting growth and/or yield.

Based upon sequence information for each mutant A-I, primers weredesigned to amplify the mutant erecta alleles from seedling materialderived from 20 seeds. Amplification is performed under standardconditions, to identify for each mutant, plants that are homozygous,heterozygous or null at the ERECTA locus. Homozygous TOS17 mutants B andE, and heterozygous lines and null lines in all lines A-I wereidentified.

In parallel to gaining information on whether or not the mutant lineswere homozygous or heterozygous or null mutants, specific plant partsare removed for analysis of the consequences of the mutations on theERECTA gene expression (levels and tissue specificity of expression),using quantitative real-time PCR as described herein. Additionally, thetranspiration efficiency phenotype of each mutant line is determined bymeasuring C&O isotopic composition and ash contents of plant samples.

Initial results on ¹³C isotopic composition of mature blades of riceseedlings reveals significant variation between mutant lines (−32.8 to−34.2 per mil) and, in at least 4 mutants, significant deviations fromthe wild type values, towards more negative values, suggesting that theerecta mutations do affect transpiration efficiency in rice, as inArabidopsis.

Similar methods as above are applied to anaylzing the progeny of themutant plants, to facilitate analysis of the effects of the erectamutations under a range of conditions, including flooding (as is themost common practice for Nipponbare), water stress such as from soildrying (upland rice growth conditions) or low air humidity (heatspells). Differences in plant morphology, anatomy and apical dominanceare noted under each environmental condition. Parameters that arecharacterised include tillering patterns, the anatomy of leaves andmeristems, development and growth rates.

Comparisons between mutants A-I are further used to characterize therole of the different protein domains in conferring different phenotypesobserved for each line under different environmental and/or agriculturalgrowth conditions. It is interesting that, among the 4 mutants thatexhibit much lower C isotopic composition than the wild type, three arethose mutants where the TOS17 insert affects the membrane spanningregion.

EXAMPLE 11 Effect of Silencing ERECTA Gene Expression on TranspirationEfficiency

To confirm the role of the ERECTA gene in conferring the transpirationefficiency phenotype on a plant, expression of the wild-type ERECTAallele is reduced or inhibited using standard procedures in plantmolecular biology, such as, for example, antisense inhibition of ERECTAexpression, or the expression of inhibitory interfering RNA (RNAi) thattargets ERECTA expression at the RNA level. All such procedures will bereadily carried out by the skilled artisan using the disclosednucleotide sequences of the ERECTA genes provided herein or sequencescomplementary thereto.

For transformation of rice and Arabidopsis, transgenes are prepared indisarmed, non-tumorigenic binary vectors carrying T-DNA left and rightborders and a selectable marker operable in E Coli.

Binary vectors used for DNA transfer include vectors selected from thegroup consisting of:

-   -   1. pPZP222 (Hajdukiewicz et al, 1994, Plant Mol Biol 25,        989-994);    -   2. PBI 121 (Clonetech) (Ueda et al 1999, Protoplasma 206,        201-206);    -   3. pOCA18 (Olszewski et al 1988, Nucl. Acid Res, 16        10765-10782);    -   4. pGreen and pSoup or variants thereof (Hellens et al., 2000,        Plant Mol Biol 42, 819-832) and    -   5. binary vectors developed on the pCAMBIA vectors backbone        described at the webiste of CAMBIA.

The starting material for all these vectors was the backbone developedby Hajdukiewicz et al., 1994. The pPZP series of vectors comprise (i) awide-host-range origin of replication from the Pseudomonas plasmid pVS1,which is stable in the absence of selection; (ii) the pBR322 origin ofreplication (pMB9-type) to allow high-yielding DNA preparations in E.coli; (iii) T-DNA left (LB) and right (RB) borders, including overdrive;and (iv) a CaMV35S promoter expression cassette. While the pPZP seriesof vectors also served as the backbones for the pCAMBIA series, theyhave been very extensively modified for particular applications.

Vectors containing in their T-DNA various combinations of the followingcomponents are particularly preferred:

-   -   1. hptII resistance gene cassette for conferring resistance to        hygromycin on transformed plant material, wherein expression of        hptII is operably under control of Ubi1 or 35S promoter;    -   2. a reporter gene cassette comprising nucleic acid encoding the        EGFP (Enhanced Green Fluorescence Protein) and/or        beta-glucuronidase (GUS and GUSPlus) reporters;    -   3. Gal4/VP16 transactivator cassette; and    -   4. one or more plant gene expression cassettes comprising either        full-length or partial cDNAs of ERECTA genes in the sense or        antisense orientation, or capable of expressing RNAi comprising        sequences derived from the ERECTA gene, including any genomic        fragments of plant DNA.

The binary vectors are transferred to disarmed strain AGL1 ofAgrobacterium tumefaciens by standard tri-parental matings (Ditta et al,1980, Proc. Natl Acad. Sci. 77,7347-7351) using the pRK2013 helperstrain of E coli. A thaliana plants are transformed using the standardfloral dip method for transformation by disarmed strains of Atumefaciens (Clough and Bent, 1998, The Plant Journal 16, 735-743). Riceis transformed by generating embryogenic calli from excised embryos andsubjecting the embryogenic calli to Agrobacterium tumefaciens mediatedtransformation according to published procedures (eg Wang et al 1997, J.Gen and Breed, 51 325-334, 1997).

Transformed plants are analyzed to confirm that those lines expressingantisense or RNAi constructs have reduced expression of functionalERECTA protein and more closely resemble the erecta phenotype than dowild-type plants or plants ectopically expressing a wild-type ERECTAgene in the sense orientation.

EXAMPLE 12 Identification of a Sorghum Ortholog of A. thaliana ERECTA

An ortholog of the A. thaliana ERECTA allele (SEQ ID NO: 1) in sorghumwas identified in silico by homology searching of the NCBI proteindatabase using the BLAST programme under standard conditions. The inputsequence was SEQ ID NO: 2. The nucleotide sequence of the sorghumortholog is presented in SEQ ID NO: 5, with the encoded proteincomprising the amino acid sequence set forth in SEQ ID NO: 6.

EXAMPLE 13 Identification of A. thaliana ERECTA Homologs

Two homologs of the A. thaliana ERECTA allele (SEQ ID NO: 1) wereidentified in silico by homology searching of the NCBI protein databaseusing the BLAST programme under standard conditions. The input sequencewas SEQ ID NO: 2. The nucleotide sequences of the A. thaliana ERECTAhomologs are presented in SEQ ID NOs: 7 and 9, with the encoded proteinscomprising the amino acid sequences set forth in SEQ ID NOs: 8 and 10,respectively.

T-DNAinsertional mutants for these two homologous genes, both on chr5,have been identified in the Salk Institute mutant collection (webaddress: signal.salk.edu/cg:-bin/tdnaexpress). Several of these mutantswere ordered: Salk_(—)007643 and Salk_(—)026292 for gene At5g07180;Salk_(—)045045 and Salk_(—)081669for gene At5g62230. Primer pairs weredesigned in order to determine insert copy number andhomozygozity/heterozygozity in the seedlings grown from the seeds thatwere received. Homozygous lines with 1 insert were identified and areunder characterisation in order to compare the expression patterns(tissue localisation and mRNA levels) of the two genes and of the ERECTAgene across a range of environmental conditions and determine whetherthe three genes are functionally related.

EXAMPLE 14 Identification of Wheat Orthologs of A. thaliana ERECTA

Partial cDNA sequence of orthologs of the A. thaliana ERECTA allele (SEQID NO: 1) in wheat were initially identified in silico by homologysearching of the NCBI protein database using the BLAST programme understandard conditions. It was necessary, however, to conduct additionalsearches of private databases in order to link the partial sequencesidentified in the NCBI database. Correction of partial sequences locatedin the NCBI database was also necessary in order to generate a contigcorresponding to the wheat ERECTA ortholog.

The input sequence is the A. thaliana (SEQ ID NO: 2) or rice (SEQ ID NO:4) amino acid sequences or a nucleotide sequence encoding same. Thenucleotide sequences of the wheat ortholog are presented in SEQ ID NOs:11-19, with the encoded proteins comprising the amino acid sequences setforth in SEQ ID NO: 20.

The sequence set forth in SEQ ID NOs: 11 to 18 are partial cDNAsequences. The corresponding sequence of the wheat ERECTA ortholog (SEQID NO: 19) is isolated by standard nucleic acid hybridization screeningof a wheat cDNA library.

To confirm the role of the wheat ERECTA orthologs in transpirationefficiency, expression data sets are used for in silico studies ofERECTA gene expression in a range of tissues of wheat plants grown undera range of environmental conditions, thereby providing indications oftissue specificities in expression patterns and preliminary data on thetypes of environments where the ERECTA ortholog is most likely to play aphysiological role in relation to water use in this species. In thesestudies, nucleic acid comprising the sequence set forth in SEQ ID NO: 11to 19, or a sequence complementary thereto, are used to producehybridization probes and/or amplification primers.

Additionally, an ERECTA gene (SEQ ID NO: 11 to 19) in the sense orantisense orientation is introduced into wheat, thereby producingtransformed expression lines. Gene constructs are specifically tosilence ERECTA gene expression using RNAi technology, or alternatively,to ectopically express the entire open reading frame of the gene.

Based upon similar function, the open reading frame of the A. thalianaERECTA gene (i.e., SEQ ID NO: 1) is also introduced into wheat plantmaterial in the sense orientation, thereby ectopically expressing A.thaliana ERECTA in wheat.

Gene constructs are introduced into wheat following any one of a numberof standard procedures, such as, for example, using A. tumefaciensmediated transformation as described in published AU 738153 or EP856,060-A1 or CA 2,230,216 to Monsanto Company, or using publishedbiolistic transformation methods as described by Pellegrineschi et al.,Genome 45(2), 421-30, 2002. Accordingly, genetic transformation isreadily used to generate wheat lines with altered expression of anERECTA gene. About 30 to 40 different transformants are produced,depending upon the efficiency of RNAi in reducing expression of ERECTAin wheat.

Primary transformants (T0) are characterized to determine the number andloci at which transgenes are inserted. T1 and T2 segregating progeniesare then generated from selected T0 transformants, and analyzed todetermine segregation ratio and to confirm the number of loci havinginserted transgenes. Those T1 and/or T2 lines having single transgeneinsertions are selected and used to generate and multiply seed forphysiological studies.

Water use efficiency in the T1 and/or T2 lines is determined through (a)gravimetric measurements of water transpired and biomass increases; (b)¹³C isotopic discrimination in plant tissues, (i.e., by determining Δ;and (c) ash content of plant tissue.

Meristem and leaf development are also analyzed, especially with respectto the differentiation and anatomy of the epidermis, the stomatalcomplexes and the mesophyll tissue and by examining leaf gas exchangeproperties. This is done using microscopy, in situ imaging techniquesand concurrent on-line measurements of C isotopic discrimination (Δ) andof CO₂ and water fluxes in and out of leaves. Information on generegulation and the network of genes in which the ERECTA orthologoperates in its effects on transpiration efficiency, is determined bytranscriptome analysis of a restricted set of the transgenic lines withaltered ERECTA expression.

As described herein for A. thaliana and rice, correlations betweenphysiological measurements and gene expression level or copy numberconfirm the role of the ortholog in conferring the transpirationefficiency phenotype in wheat.

EXAMPLE 15 Identification of a Maize Ortholog of A. thaliana ERECTA

Partial cDNA sequence of ortholog of the A. thaliana ERECTA allele (SEQID NO: 1) in maize were initially identified in silico by homologysearching of the NCBI protein database using the BLAST programme understandard conditions. It was necessary, however, to conduct additionalsearches of private databases in order to link the partial sequencesidentified in the NCBI database. Correction of partial sequences locatedin the NCBI database was also necessary in order to generate a contigcorresponding to the maize ERECTA ortholog.

The input sequence was SEQ ID NO: 2. The nucleotide sequence of a maizeortholog is presented in SEQ ID NOs: 21 to 44, with the encoded proteincomprising the amino acid sequence set forth in SEQ ID NO: 45.

The sequence set forth in SEQ ID NOs: 21 to 43 are partial cDNAsequences. The corresponding sequence of the maize ortholog (SEQ ID NO:44) is isolated by standard nucleic acid hybridization screening of awheat cDNA library.

To confirm the role of the maize ERECTA ortholog in transpirationefficiency, expression data sets are used for in silico studies ofERECTA gene expression in a range of tissues of maize plants grown undera range of environmental conditions, thereby providing indications oftissue specificities in expression patterns and preliminary data on thetypes of environments where the ERECTA ortholog is most likely to play aphysiological role in relation to water use in this species. In thesestudies, nucleic acid comprising the sequence set forth in SEQ ID NO:15, or a sequence complementary thereto, is used to producehybridization probes and/or amplification primers.

Additionally, collections of transposon-tagged maize mutants aresearched to select those having insertions that affect expression of theERECTA gene and the expression level and/or copy number of the ERECTAortholog is correlated to transpiration efficiency under the range ofenvironmental growth conditions, essentially as described herein for A.thaliana and rice.

Additionally, an ERECTA gene in the sense or antisense orientation isintroduced into maize, thereby producing transformed expression lines.Gene constructs are specifically to silence ERECTA gene expression usingRNAi technology, or alternatively, to ectopically express the entireopen reading frame of the gene.

Based upon similar function, the open reading frame of the A. thalianaERECTA gene (i.e., SEQ ID NO: 1) is also introduced into maize plantmaterial in the sense orientation, thereby ectopically expressing A.thaliana ERECTA in maize.

Gene constructs are introduced into maize following any one of a numberof standard procedures, such as, for example, any of the methodsdescribed by Gordon-Kamm et al., Plant Cell 2(7), 603-618, 1990; U.S.Pat. No. 5,177,010 to University of Toledo; U.S. Pat. No. 5,981,840 toPioneer Hi-Bred; or published US application No. 20020002711 A1 (Goldmanand Graves);. Accordingly, genetic transformation is used to generatemaize lines with altered expression of an ERECTA gene.

About 30 to 40 different transformants are produced, depending upon theefficiency of RNAi in reducing expression of ERECTA.

Primary transformants (T0) are characterized to determine the number andloci at which transgenes are inserted. T1 and T2 segregating progeniesare then generated from selected T0 transformants, and analyzed todetermine segregation ratio and to confirm of number of loci havinginserted transgenes. Those T1 and/or T2 lines having single transgeneinsertions are selected and used to generate and multiply seed forphysiological studies.

Water use efficiency in the T1 and/or T2 lines is determined through (a)gravimetric measurements of water transpired and biomass increases; (b)¹³C isotopic discrimination in plant tissues, (i.e., by determining Δ);and (c) ash content of plant tissue.

Meristem and leaf development are also analyzed, especially with respectto the differentiation and anatomy of the epidermis, the stomatalcomplexes and the mesophyll tissue and by examining leaf gas exchangeproperties. This is done using microscopy, in situ imaging techniquesand concurrent on-line measurements of Δ and of CO₂ and water fluxes inand out of leaves. Information on gene regulation and the network ofgenes in which the ERECTA ortholog operates in its effects ontranspiration efficiency, is determined by transcriptome analysis of arestricted set of the transgenic lines with altered ERECTA expression.

As described herein for A. thaliana and rice, correlations betweenphysiological measurements and gene expression level or copy numberconfirm the role of the ortholog in conferring the transpirationefficiency phenotype in maize.

EXAMPLE 16 Mechanism of Enhanced Transpiration Efficiency andInheritance of ERECTA in Arabidopsis (Landsberg and Columbiabackgrounds)

The present inventors performed direct measurements of transpirationefficiency (ratio of CO₂ assimilation rate to transpiration rate) inboth Landsberg and Columbia backgrounds. To confirm the role of ERECTAunder a wider range of agronomically relevant conditions, thetranspiration efficiencies of transformed plants carrying an ERECTAallele in response to varying environmental conditions (i.e., soil waterand ion content, atmospheric humidity and CO₂ levels) were determinedand compared to the response of wild type plants (e.g., ER). Results ofthese experiments are presented in FIGS. 6-11, and Tables 8 and 9.

Data in FIG. 6 show that the enhanced transpiration efficiency obtainedby inserting a transgene carrying the wild type ER allele in the Ld-er1mutant (line T2+ER) is mostly due to a decreased stomatal conductance.The phenotype of the transgenic line (T2+ER in graphs) is similar tothat of a Ld-ER ecotype near isogenic to Ld-er1 obtained from the StockCentre (line 3177 on graphs). The increased transpiration efficiency intransgenic ER, compared to levels observed in wild type ER line isobserved under both current ambient CO₂ levels and increased CO₂ levelsthat are within the limits predicted to occur worldwide over the nexttwo decades.

Data in FIG. 8 show that the reduced stomatal conductance in the ER T2transgenic line compared to the Ld-er1 line is, at least for a largepart, caused by a reduced stomatal density (decrease in the number ofstomata per unit area by more than half, down to similar levels as thoseobserved in wild type Ld-ER). This decrease in stomatal density isrelatively higher than that in the density of epidermal cells whosesurface area is increased by only about 10%. It therefore follows thatthe ER transgene has affected stomatal development, specifically, andcaused a decreased in stomatal index. These data show complementationwith respect to the processes driving variation in transpirationefficiency.

Reciprocal crosses were also performed between the two parental linesNW20 (Ld-er1) and Col4 (Col-ER). The notation F1 (Col*Ld) refers to theF1 plants where Col was the recipient of Ld pollen, while the notationF1 (Ld*Col) indicates the converse (Ld ovary receiving Col pollen).Initial analysis of these two types of F1 plants has been made for: gasexchange and photosynthetic properties, transpiration efficiency (FIG.7) and C isotopic composition (Table 9), rosette shape and developmentalrate, anatomy of leaf epidermis (FIG. 8), flowering date, inflorescenceand pod shape. Consistent with our analysis of complementationexperiments, the data show that the ERECTA gene affects all thesephenotypes and not only inflorescence and pod shape.

The data also show a complex inheritance of the ERECTA gene, such thatthe gene is dominant, with no reciprocal effect on pod shape (longerpods, longer stems and pedicels in all F₁ plants, similar to the Col-ERparent). However, for other traits, results indicate maternal effects:hence the transpiration efficiency values (see FIG. 7 a) and rosettescarbon isotope composition in F₁ plants (Table 9) are intermediatebetween the parental values, but different between the two sets of F1plants: values for F₁ plants (Col*Ld) are closer to the Col values,while those for F1 plants (Ld*Col) are closer to values for the Ldparent.

Data in FIG. 8 indicate that stomatal conductance (transpiration perunit leaf area, FIG. 8 a) displays values close to the Ld-er1 parent inall F1 plants, despite the stomatal densities being close to the Col-ERparent (FIG. 8 c). This shows that the ER gene affects not onlyepidermis development but also stomatal aperture (dynamics of stomata)and that while the ER effect on stomatal density appears to be dominant,effect on stomatal aperture is not.

Data in FIG. 9 show the effect of various er mutations (in Colbackground, mutants obtained from the Stock Centre or Dr Torii) on thenumber of stomata per unit leaf area. The stomatal densities for all buttwo of those mutants are greater than those the ColER wild type leaves,and confirm the effect of the ERECTA gene on that parameter.

Data in FIG. 10 show that enhanced transpiration efficiency in the ERtransgenic line compared with null Ld-er1 (no insertion of transgene) isconfirmed by the less negative C isotopic composition values measured inleaf material (compare values for lines NW20 and CS20 (Ld er1; lines 16and 17 on x-axis) and a transgenic T2 1d-ER line, homozygous for he ERtransgene (line 19 in the Figure). The C isotopic values measured in theER transgenic line are similar to those in the near isogenic Ld-ERecotype (line 18 in FIG. 10). This demonstrates complementation on thisphenotypic trait, and validates once again the use of C isotopiccomposition as a quantitative indicator (substitute) of transpirationefficiency.

Data in FIG. 10 also the C isotopic compositions of a range of Col-ermutants, including those analysed in FIG. 9 for stomatal densities. Mostmutants show more negative C isotopic values than the COL-ER ecotype.This is consistent with the increased stoamtal densities described inFIG. 9 and with all other comparisons of C isotopic compositions ordirect measurements of transpiration efficiencies in er/ER lines andagain indicative of the positive effect of the ER allele ontranspiration efficiency.

A few mutants in FIG. 10 stand out, eg Col-er105, or line 3140 (a linefrom NASC carrying the er1 and gl1-1 mutations). As genetic informationis available for these mutants (nature and position of mutations) thesemutants provide very useful functional information on the proteindomain(s) of the ERECTA protein that are essential for conferring thetranspiration efficiency phenotype and underlying processes.

The present inventors also perform direct measurements of transpirationefficiency (ratio of CO₂ assimilation rate to transpiration rate) inseveral T2 transformants generated in a Columbia background (i.e.transformation of mutant er-105 and er-2/106 above). Results from thesemeasurements are shown in FIG. 11. These data show that the phenotypecan be complemented in a Columbia background, as determined by measuringtranspiration efficiency, transpiration and CO₂ assimilation rates.Complementation is observed under conditions of both high humidity andlow humidity, hence the demonstration that the ERECTA gene plays a rolein the control of transpiration efficiency under both well watered anddrought conditions, and that overexpression of that gene has thepotential of increasing growth and resistance to drought and droughtrelated stresses.

More particularly, the data in FIG. 11 demonstrate the role of theERECTA gene on transpiration efficiency across a range of humidities,including low humidities such as prevail in warm and dry areas:

-   -   the er-105 mutant which carries a knock-out mutation of ERECTA        (quasi no ER transcript) (open black squares) in Col0 background        has lower transpiration efficiency than the wild type near        isogenic Col0 (open triangles).    -   this mutant was transformed with an ER transgene under the 35S        promoter and several ER homozygous T2 lines were produced (solid        circles). Those lines (5 independent transformants are included        in the graph) have much increased transpiration efficiencies        (+40 to 70%) compared to the null lines (solid squares) and        similar to those measured for the wild type ER-Col0 line, across        the whole range of leaf to air vapour pressure deficit tested In        our experiments.

Additionally, null lines that carry no transgene insertion but wentthrough transformation and selection on antibiotics display similarvalues as the starting er-105 mutant demonstrating that thesemanipulations themselves have no detectable confounding effect ontranspiration efficiency. TABLE 1 QTL Analysis of Carbon IsotopeDiscrimination in Lister and Dean's Recombinant Inbred Lines RUN No.chr2 locus QTL chr4 locus CONCLUSION Experimental conditions (cM)analysis method (cM) QTLs number predicted map position Run 1 (40 lines)Glasshouse- 12 h day length 58.5 SIM&CIM 2 chr2:  58.5-61.02irradiance150-350 μE m⁻² s⁻¹ 46.77 SIM&CIM chr2: 46.77-50.75 Seedlingstransferred from 61.02 SIM 108.5 agar plates Run1 data 56.94 to 58.00CIM&SIM 1 but with using different 46.77 to 50.75 SIM markers 63.02 Run1  58.5 to 61.02 2 with different number of 56-61 lines Run2 GlasshouseSeptember from seeds sown on soil batch 1 50.75 CIM (QTL cart) 2 chr2:56.94-61.02 61.02 MQTL chr2: 50.75 batch 2 ?50.75 MQTL NS batch 3-5 allbatches 58.5 MQTLcart NS 56.94-58.5  MQTL Run 3 37 lines: parents andlines with crossing-overs on chromosome 2 5 growth conditions differingin humidity, irradiance, mode of establishment (seeds sown on soil orseedlings transplanted from agar) batch B 61.02-61.06 108 NS batch C56.94-58.00 batch D 63.02 QTLcar 63.02 MQTL all batches (conditions)58.5 1 or 2 chr2: 56.94-58.5  61.02 3 chr2: 61.02-63.02 Run 4 50.74chr2: 50.74 same lines as Run 3 growth chambers 10 h daylight Run 550.74 1 chr2: 50.74 repeat of run 1 BUT ALL lines Run 7 same lines as inrun 1 but in growth chamber and higher light 10 h daylength 46.77-50.75CIM&SIM 1 chr 2: 46.77-5065  470-510 μE m⁻² s⁻¹ irradiance

TABLE 2 Isogenic ER line and Background Mutation Stock Centre name StockCentre Name Landsberg er1 CS20 or NW20^(a) 3177 or CS163 Columbiaer2^(b)/er106 3401 Col1 or 3176 Columbia er105^(c) Col3 with gl1 markeror Col0^(a)NW20 is an Ler parent for Lister and Dean's recombinant lines,carrying the er1 mutation. Lines 3177 or CS163 are the closest isogenicER lines.^(b)er2 is an er allele identified by Rédéi in Col background. Col1 or3176 are the closest Col near-isogenic lines. The er2 is same mutationas mutation er-106 later reported by Torii and collaborators (Lease etal. 2001)^(c)er105 was isolated from a fast-neutron-irradiated Col seedpopulation (Torii et al., 1996).d, Col4, the Col parent for the Lister and Dean's parent wassystamically included in all comparisons.

TABLE 3 Comparison of er/ER lines in both Col and Ld background forcarbon isotope discrimination values (per mil) in leaf material under arange of environmental conditions Differences in mean carbon isotopediscrimination values (per mil) (7) er1-Col4 (1) (parental Run er-ER (2)(3) (4) (5) (6) lines for (8) (9) No. (all lines) er105-Col0 er105-Col4er105-3176 er2-3176 er1-3177 RILs) er105-Coli er1-Coli 1 0.13 0.16 0.162 0.89 1.18 1.18 3 0.26 0.11 0.11 4 1.12 1.60 1.60 5 1.03 1.83 1.67 0.920.64 0.82 1.75 0.73 6 0.70 1.13 1.01 0.71 0.27 0.74 0.73 0.95 0.73 70.70 1.32 1.12 1.23 0.75 0.35 0.05 1.22 0.15 8 0.59 1.16 1.11 1.19 0.540.28 0.06 1.16 0.17 9 0.30 1.09 0.77 0.77 0.02 0.00 0.56 0.88 0.28 10 0.56 1.05 0.94 0.87 0.38 0.39 0.33 0.95 0.36 (1) difference er-ER (2)(3) (4) (5) (6) (7) (8) (9) Run # (all lines) er105-Col0 er105-Col4er105-3176 er2-3176 er1-3177 er1-Col4 er105-Coli er1-Coli 11  0.48 0.400.52 0.40 0.52 12  0.36 0.82 1.31 1.08 0.33 0.05 0.07 1.07 0.01 13  0.380.90 0.82 0.07 0.60 0.52 0.86 0.56 14  0.65 1.42 0.60 0.58 0.06 1.010.32 15  0.82 0.82 0.82 For all runs: Mean 0.60 1.01 1.00 1.04 1.41 0.400.41 0.95 0.42 S.E. 0.07 0.14 0.11 0.11 0.10 0.08 0.09 0.12 0.08 ForCommon runs: Mean: 0.58 1.10 1.12 1.04 0.41 0.38 0.39 1.11 0.37 S.E.0.08 0.05 0.11 0.11 0.10 0.09 0.10 0.10 0.09

TABLE 4 Run 9 - December 2001: Leaf gas exchange measurements in er/ERArabidopsis lines (4) (1) (2) (3) A/E (5) (6) E A Gw (mmolC/ pa pi (7)(8) Genotype (mmolH₂O/m²/s) (μmolC/m²/s) (mol/m²/s) molH₂O) (μbar)(μbar) pi/pa 1 − pi/pa Row Ld-ER 3177 Mean 3.38 12.33 0.273 3.67 360 2820.782 0.218 (1) S.E. 0.48 1.64 0.039 0.14 10 11 0.010 0.010 Row Ld-erNW20 Mean 2.59 8.73 0.218 3.38 348 280 0.804 0.196 (2) S.E. 0.07 0.310.005 0.04 5 4 0.002 0.002 Row Col-ER 933 Mean 3.41 13.55 0.291 4.06 350270 0.772 0.228 (3) S.E. 0.40 1.16 0.040 0.22 4 7 0.020 0.020 Row Col-ER3176 Mean 2.23 10.13 0.180 4.55 346 254 0.734 0.266 (4) (Col1) S.E. 0.501.47 0.048 0.24 5 9 0.021 0.021 Row Col-er er105 Mean 2.27 8.55 0.1983.76 356 283 0.795 0.205 (5) S.E. 0.03 0.17 0.005 0.07 11 10 0.006 0.006Row Col-er er2 Mean 3.06 11.90 0.256 3.92 357 279 0.780 0.220 (6) S.E.0.22 0.56 0.027 0.12 1 6 0.014 0.014 CONCLUSION: ComparisonLd-ER/Ld-erer line has lower A/E with lower g and lower A. The difference in A/E isdriven by A Comparison 933/NW20 NSW20 (er1) has lower A/E with lower gand lower A Comparison Col1/Ld-er1 The difference in A/E is driven by AComparison Col1/Col-er105 er105 has MUCH lower A/E with Higher g andlower A i.e. the difference in A/E is driven by A and g ComparisonCol1/Col-er2 er2 has lower A/E with MUCH higher g and HIGHER A i.e. thedifference in A/E is driven by g and is opposed or not driven by ANOTE:p_(a) and p_(I) are the ambient and intercellular partial pressures ofCO₂, respectively.

TABLE 5 QTL position experimental run LOD P(0.05) P(0.01) cM gap LOD −LOD_(P0.05) R² Total R²  1 5.2861 4.7469 5.9923 39.32-50.65 0.5392 0.190.89  4 3.519 3.3561 4.2134 50.63-50.65 0.1629 0.29 0.57  7 9.64893.9627 4.9083 50.63-50.65 5.6862 0.53 0.77  9 6.1748 4.2328 5.027846.77-50.65 1.942 0.44 0.81 10 Zero 3.6051 4.3889 Qtl's 16 11.5132 3.1883.8896 48.96-50.65 8.3252 0.64 0.65  2(batch1) 5.5459 3.3264 4.290750.63-51.02 2.2195 0.26 0.40LOD = log₁₀ of the likelihood ratio

TABLE 6 Summary table of the lines used for initial functionalcharacterisation and analysis of ER effects: Background Stable T2homozygous ecotype ER transformants Null er control Col-er105 T8; T29;T19; T61 T18 Col-er106/er2/3401 T165; T169; T279; T290 T143 Ld-er1 T3-7KNW20

TABLE 7 Carbon isotope composition (per mil) of 3 mature leaves, groundtogether harvested 4/6/03 ie 32 days after sowing from still vegetativerosettes Null background T2 ER homozygous transgenics er transformants(er) mutant Line: T46 T29 T18 Col-er105 −31.4 −31.2 −32.2 Col er106/er2/Line: T145 T165 T279 T290 T154 T143 T247 3401 −30.4 −31 −30.5 −30.8−31.5 −32 −31.7 Average: −30.6 −31.7 −31.7 se: 0.15 0.12 Ld-er1 lineT3-7K NW20 −30.4 −31.3

TABLE 8 C isotopic composition (per mil) Erecta Line Run 14 Run 18alleleLine name Average St Err Average St Err Col_er 102K −29.4 .011mutants 103K −29.0 .10 105C −31.4 0.12 −30.3 .09 105KH −30.2 −29.3 0.11105KS −29.8 0.07 −29.5 .07 3401 −30 0.21 −29.4 .05 106C −30.2 0.04 −29.4.12 108K −30.2 0.07 −29.5 .06 111KH −30.4 0 −29.5 .11 111KS −30.2 0.11−29.7 .12 114K −30.2 0.11 −29.5 .04 116K −29.7 0.21 −29.0 .13 117K −29.70.18 −29.3 .08 3140 −32.2 .06 Col0_ER 1093 −29.6 0 −29.0 .10 Ld_er1 NW20−30.0 0.25 −28.9 .15 CS20 −29.9 0.08 Ld_ER 3177 −29.4 −28.2 .09Transgenic Ld_er1 + wild 3-7K −29.5 0.07 −28.4 .10 type ER

TABLE 9 C isotope composition (per mil) Col_ER (line 933) −28.1 F1(Col * Ld) −28.5 F1 (Ld * COL) −29.3 Ld-er1 (line NW20) −29.9

1. A method of selecting a plant having enhanced transpirationefficiency, comprising detecting a genetic marker for transpirationefficiency which marker comprises a nucleotide sequence linkedgenetically to an ERECTA locus in the genome of the plant and selectinga plant that comprises or expresses the genetic marker.
 2. The methodaccording to claim 1 wherein the genetic marker comprises an ERECTAallele or erecta allele, or a protein-encoding portion thereof.
 3. Themethod according to claim 2 wherein the genetic marker comprises anucleotide sequence having at least about 55% overall sequence identityto at least about 20 nucleotides in length of any one of SEQ ID Nos: 1,3, 5, 7, 9, 11 to 19 or 21 to 44 or a complementary sequence thereto. 4.The method according to claim 2 wherein the genetic marker comprises anucleotide sequence selected from the group consisting of: (a) asequence having at least about 55% identity to a sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ IDNO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQID NO: 19 SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 SEQ ID NO: 33, SEQ IDNO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38; SEQID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43and SEQ ID NO: 44; (b) a sequence encoding an amino acid sequence havingat least about 55% identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 20 and SEQ ID NO: 45; and(c) a sequence complementary to (a) or (b).
 5. The method according toclaim 1 wherein the plant is selected from the group consisting ofArabidopsis thaliana, rice, sorghum, wheat and maize.
 6. The methodaccording to claim 1 comprising linking the transpiration efficiencyphenotype of the plant to the expression of the marker in the plant. 7.The method according to claim 1 comprising linking a structuralpolymorphism in DNA to a transpiration efficiency phenotype in theplant.
 8. The method according to claim 7 wherein the polymorphism isdetermined by a process comprising detecting a restriction fragmentlength polymorphism (RFLP), amplified fragment length polymorphism(AFLP), single strand chain polymorphism (SSCP) or microsatelliteanalysis.
 9. The method according to claim 1 comprising hybridizing aprobe or primer of at least about 20 nucleotides in length from any oneof SEQ ID Nos: 1, 3, 5, 7, 9, 11 to 19 or 21 to 44 or a complementarysequence thereto to genomic DNA from the plant, and detecting thehybridization using a detection means.
 10. The method according to claim1 wherein the selected plant has enhanced transpiration efficiencycompared to a near-isogenic plant that does not comprise or express thegenetic marker.
 11. A method of selecting a plant having enhancedtranspiration efficiency, comprising: (a) screening mutant ornear-isogenic or recombinant inbred lines of plants to segregate allelesat an ERECTA locus; (b) identifying a polymorphic marker linked to saidERECTA locus; and (c) selecting a plant that comprises or expresses themarker.
 12. A method of modulating the transpiration efficiency of aplant comprising introducing an isolated ERECTA gene or an allelicvariant thereof or the protein-encoding region thereof to a plant andselecting a plant having a different transpiration efficiency comparedto a near-isogenic plant that does not comprise the introduced ERECTAgene or allelic variant or protein-encoding region.
 13. The methodaccording to claim 12 wherein the ERECTA gene or allelic variant orprotein-encoding region comprises a nucleotide sequence selected fromthe group consisting of: (a) a sequence having at least about 55%identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 21, SEQID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32 SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ IDNO: 36, SEQ ID NO: 37, SEQ ID NO: 38; SEQ ID NO: 39, SEQ ID NO: 40, SEQID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44; and (b) asequence encoding an amino acid sequence having at least about 55%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 20 and SEQ ID NO:
 45. 14. The method accordingto claim 12 wherein the plant is selected from the group consisting ofArabidopsis thaliana, rice, sorghum, wheat and maize.
 15. The methodaccording to claim 12 wherein the ERECTA gene or allelic variant orprotein-encoding region is introduced to the plant by a processcomprising introgression.
 16. The method according to any one of claim12 wherein the ERECTA gene or allelic variant or protein-encoding regionis introduced to the plant by a process comprising transforming plantmaterial with a gene construct comprising the gene or allelic variant orprotein-encoding region thereof.
 17. The method according to claim 12further comprising expressing the introduced gene or allelic variant orprotein encoding region in the plant.
 18. The method according to claim12 wherein transpiration efficiency is enhanced in the plant.
 19. Themethod of claim 18 wherein the transpiration efficiency is enhanced as aconsequence of the ectopic expression of an ERECTA allele or theprotein-encoding region thereof in the plant.
 20. The method accordingto claim 12 wherein transpiration efficiency is reduced in the plant.21. The method of claim 20 wherein the transpiration efficiency isreduced as a consequence of reduced expression of an ERECTA allele inthe plant.
 22. A plant having modified transpiration efficiency comparedto a near-isogenic plant wherein said plant is produced by a processcomprising performing the method according to claim
 12. 23. The plant ofclaim 22 selected from the group consisting of a rice plant, a wheatplant and a maize plant.
 24. An isolated ERECTA gene from wheat capableof determining or modulating the transpiration efficiency of a plantwherein said isolated ERECTA gene comprises a nucleotide sequenceselected from the group consisting of: (i) a sequence selected from thegroup consisting of: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ IDNO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, andSEQ ID NO: 19; (ii) a sequence encoding the amino acid sequence setforth in SEQ ID NO: 20; and (iii) a sequence that is complementary to(i) or (ii).
 25. An isolated ERECTA gene from maize capable ofdetermining or modulating the transpiration efficiency of a plantwherein said isolated ERECTA gene comprises a nucleotide sequenceselected from the group consisting of: (i) a sequence selected from thegroup consisting of: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 29, SEQ ID NO:
 30. SEQ ID NO: 31, SEQ ID NO: 32 SEQ ID NO: 33,SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:38; SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ IDNO: 43 and SEQ ID NO: 44; (ii) a sequence encoding the amino acidsequence set forth in SEQ ID NO: 45; and (iii) a sequence that iscomplementary to (i) or (ii). 26-36. (canceled)